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OURSE
ESCRIPTION
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RGANIZATION
AJOR
OURSE
BJECTIVES
SSESSMENT
EPARTMENT WEB SITE
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UTLINES
ECTURES
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Welcome to the Department of Clinical Biochemistry. The aim of this course guide is to provide you with clear description of the course objectives, contents of each topic together with its lectures, tutorials and practicals, which are presented in a sequential manner. Also it states clearly what is expected from you to achieve together with the evaluation procedures. There is no reason to suppose that Biochemistry is intrinsically uninteresting, difficult to understand or an obstacle to be overcome during your progress to a professional qualifications. In the contrary you can enjoy its study if you remember the simple biological principals that the body is formed from organs and tissues and these are formed from aggregates of cells. The cell is build up from sub-cellular structures which by itself is an aggregate of molecules. Every human actively, like walking, breathing and even thinking are the result of the interactions of these molecules. Defective molecular processes result in disease state. Moreover Biochemistry has become a background subject for a great family of medical sciences. Understanding
Biochemistry will build for you a good foundation for understanding disease states, their prevention, diagnosis and treatment. We advice you to read the objectives of each topic before the start of its lectures and focus your attention on the important points. Take another look after the end of the lecture to make sure that these points have been understood.
Dr. Mohamed Aly Ajb Noor
Chairman, Department of Clinical Biochemistry
3

OURSE
ESCRIPTION
ND
RGANIZATION
The aim of Clinical Biochemistry course for second year student is to introduce you to the basic concepts of structure and metabolism in relation to the mechanisms by which primary foodstuffs, proteins, carbohydrates and lipids, are manipulated by the body in order to provide energy and to allow for biosynthesis of cellular material. Also you will have a good view of storage, transmission and expression of genetic information. Clinically relevant examples will be discussed.
Biochemical basis of medicine course includes and covers the following topics: allosteric proteins, enzymes, membrane and transport, bioenergetics and oxidative metabolism, anaerobic metabolism, tricarboxylic acid cycle, carbohydrate storage and synthesis in liver and muscle, oxidative metabolism of lipid in liver and muscle, biosynthesis and storage of fatty acids, lipid and lipoproteins, and cholesterol metabolism, Protein metabolism, Nucleotide and
Nucleic Acid metabolism.
The course consists of lectures, practical classes and tutorials.
Core Course Code/No Course Units
Lectures Practical Tutorial
46 15 15 Biochemistry For
Second Year
Credit Hours
5
4

AJOR
OURSE
BJECTIVES
On completion of the course in Clinical Biochemistry I student will:
1. Evaluate the biochemical logic of the human cell.
2. Explain the maintenance of cellular life in the term of energy production and biosynthetic ability.
3. Apply biochemical knowledge to solve problems of human health and disease.
4. Practice basic skills in applying laboratory techniques encountered in hospital's clinical biochemistry laboratories.
5. Appreciate the functional role of membrane proteins as pumps, gates and channels
5

STUDY STRATEGIES AND CLASS PARTICIPATION EXPECTATIONS
The course offered to 2nd year medical students in clinical biochemistry consists of scheduled lectures, tutorials and practicals which ensure smooth flow of the scientific material, in a controlled manner, through several pathways to achieve our objectives. There is some suggestion for optimal utilization of these classes by the students.
A. Lectures : The aim of the lecture is not to give information but to frame up the subject, pointing out its relation to other parts of the course, its relevance to clinical situations and to explain difficult points. To prepare your self for the lecture:
1. Give a thorough look to the course objectives and the topic outline delivered by the department and try to read the topic from the recommended textbook.
2. Note taking in lectures keeps the student in track during studying the subject.
3. If is recommended to study the topic of the lecture, if possible, in the same day, to prevent fading of knowledge. You can utilize different ways of self-testing in order to assess your grasping of the subject.
B. Practicals : For optimal utilization of the practical class time it is advisable to:
1. Read your practical worksheet so as to have view of what is expected from you to perform, observe and draw conclusions on your practical work.
2. A record of the practicals should be used according to instructions.
3. The practical time can also be used for discussing difficult theoretical or practical points with the instructor.
C. Tutorials: For optimal benefit of the tutorial, the tutorial will be reserved for open discussion about the subjects listed in the tutorial schedule. The students will be assigned these topics and will be asked to present them and be ready to change the most recent knowledge about these topics and how to defend their thoughts on scientific bases
6

The main instructional material includes lectures, practicals to steamline the applied and clinical aspects of the lectures and tutorials session to stimulate the students to participate in the teaching/learning activities.
1. Required Texts And Resources a. Lippincott Illustrated Reviews, 3rd edition, Champe & Harvey b. Baynes, Medical Biochemistry, Mosby, London.
2. Supplementary Texts And Resources a. Thomas M. Devlin, Textbook of Biochemistry with Clinical Correlations , Jon
Willey & sons, New York. b. Lubert Stryer, Biochemistry , W.H. Freeman and Company, San Francisco. c. Pamela C. Champe, Biochemistry , Lippincott Raven.
7

SSESSMENT
1.
Formative:
This form of assessment is designed to give you feedback to help you to identify areas for improvement. It includes a mixture of MCQs, short answer-questions (SAQs), extended matching questions (EMQs), problems-solving exercises and independent learning activities in all subjects. These will be given during tutorial sessions and practicals. The
Answers are presented and discussed immediately with you after the assessment. The results will be made available to you.
2.
Summative
This type of assessment is used for judgment or decisions to be made about your performance.
It serves as: a.
Verification of achievement for the student satisfying requirement b.
Motivation of the student to maintain or improve performance c.
Certification of performance d.
Grades
In this Course your performance will be assessed according to the following :
1.
Continuous Assessment 20 Marks
2.
Mid- term exam 20 Marks
3.
OSPE 20 Marks
4.
Final End of Semester Exam (Three Hours) 40 Marks
Total = 100 Marks
8

85 - 100
75 - 84
65 - 74
60 - 64
Less than 60 will be assigned as follows :
A
B
C
D
F
Excellent
Very good
Good
Pass
Fail
All grades
Exams
:
Exams will include short answer and multiple choice questions (MCQs). They will cover material presented in lecture, readings, and discussion. All exams must be taken on the date scheduled. In case of an emergency, the coordinator must be notified. No make-up exams will be provided if you fail to notify and discuss your situation with the coordinator.
Note : We will be making the journey from "womb to tomb" in 15 weeks. Therefore, this course requires an intensive coursework load. Class attendance and participation are extremely important to your learning and as such are considered in the evaluation of your course grade.
This course is recommended for students that can make the required time and energy commitment. If there is anything that the coordinator can do to assist you during the course, please feel free to contact him.
9

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
BIOCHEMICAL BASIS OF MEDICINE
Introduction: The role of clinical biochemistry in medicine & Water biochemistry
1
5 Proteins structure functions relationship
Catalytic proteins – Enzymes 6
Membrane and transport 1
1 Carbohydrate chemistry
Introduction to Metabolism, Bioenergetics , and the
Role of ATP
Glycolysis
The tricarboxylic acid cycle
Gluconeogenesis
Pentose phosphate pathway
Metabolism of Monosaccharides and Disaccharides
Lipid chemistry
Fatty acid oxidation
2
1
1
1
1
1
2
2
Fatty acid synthesis
Ketone bodies metabolism
1
1
Phospholipids metabolism 1
Disposal of amino acid nitrogen
Urea cycle and detoxification of ammonia
1
1
Individual amino acid catabolism (glycine metabolism)
Individual amino acid catabolism
(Phenylalanine and tyrosine)
1
1
10

33.
34.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
21.
Individual amino acid catabolism (Tryptophan, aspartic acid , glutamic acid, alanine and serine) and (sulfur containing amino acids)
Individual amino acid catabolism (sulfur containing amino acids)
1
1
Nucloetides Structure and Functions 1
Purine Nucleotide Metabolism 1
Pyrimidine Nucleotide Metabolism 1
Structure of Nucleic Acids 1
Organization of eukaryotic chromatin 1
DNA sequence and function 1
Replication of DNA (DNA Synthesis) 1
DNA Repair 1
Structure of Ribonucleic Acid (RNA) 1
Transcription of DNA (RNA Synthesis) and
Posttranscriptional processing
1 and
Regulation of Gene Expression
Translation of RNA (Protein Synthesis) 1
Recombinant DNA Techniques 1
46
11

The following is a list of the faculty members and staff of the Department of Clinical
Biochemistry. Students are welcome to contact any of the members of the department to answer any of their inquiries.
Name/Status
Room
No
Phone No
Prof. Mohammed Ali
Ajabnoor
B.Pharm.,M.S., Ph.D.
2/942
22-100, 22-
174
Professor
Chairman
Prof. Adil Abdel
Rafee
M.B., B.ch.,
D.M.Sc.,Ph.D.
Professor
Prof. Mohammed
Saleh Ardawi
B.Med. Sc., M.A.,
Ph.D.
Professor
Prof. Zohair M. H.
Marzouki
B.Pharm., M.Sc.,
Ph.D.
Professor
2/946
2/924
2/925
G/640
2/853
22-112, 22-
194
22-102, 22-
173
22-103, 22-
177
51-701
22-099, 22-
170
E-Mail Address md-ajaboor@yahoo.com adlirafee@yahoo.com ardawims@yahoo.com zmarzouki@ Kau .edu .sa
Prof. Abdulwahab A.
Noorwali
B. Phar., Ph.D.
Professor
Prof. Mohammed
Zelai A. Abdu
B.Sc., M.Sc., Ph.D.
Professor
2/911
22-097, 22-
172
2/852
22-098, 22-
169
Prof. Mamdouh
Youssef Souaida
M.B.ch., M.Sc.,Ph.D.
2/940 22-128
Professor
Dr. Zainy M. A. 2/945 51-048, 22anoorwaIi@wamy.org mzailaie@kaau.edu.sa drmamdouhyoussef@yahoo.com zbanjar@kaau.edu.sa
Office Hours
Sat, Mon, Wed
1.00-2.00
Daily
12.00-1.00
Daily
12.00-1.00
Dean Off., Faculty of
Pharmacy
Sat, Sun, Mon
1.00-2.00
Sat, Sun, Mon
1.00-2.00
12

Banjar 116 osama@osamagaber.com ogaber@kau.edu.sa
Dr Osama Abdul
Aziz Gaber
2/766 22-096
WWW.kaau.edu.sa/ogaber
M.B., B.ch., M.D.
Associate Professor
Dr Mohammed A
Hassanien
M.B., B.ch., M.D.
Assistant Professor
Dr. Mohammed
Shoaib Jarullah
B.Sc., M.Sc., Ph.D.
Technician
Mr. Ahmad Al-
Shamrani
B.Sc.
Technician www.osamagaber.com
2/941 22-118 mohammedhassanien700@hotmail.com
WWW.kaau.edu.sa/mhassanien mshoaibj@yahoo.com 2/912 22-130
Lab
22-120, 22-
121
2/922 22-117
Lab
22-120, 22-
121 alshamrani1@hotmail.com
Daily
11-12
13

Name/Status
Room
No
Phone No E-Mail Address Office Hours
Prof Suhad Matoug
Bahijri
B.Sc.,M.Sc., Ph.D.
Professor
Prof Enayat Mohd.
Hashem
M.B., B.ch., h.D.
Professor
Dr Amina Mohd. Al-
Ghareeb
M.B., B.ch., h.D.
Associate Professor
Dr Huda Gad
M.B., B.ch., h.D.
Assistant Professor
Dr. Eman Mokbel
Alissa ( BSc MSc PhD)
Assistant Professor
2/622 23-601
2/618 23-437 enayathasaem@hotmail.com
2/617 23-431 amina_1959@hotmail.com
2/613 23-433
2/621 23-432 biochemsme@yahoo.com drhodagad@yahoo.com ealissa@kau.edu.sa
Webpage: www.kau.edu.sa/ealissa alfayez_ff@hotmail.co.uk
(
Dr. Fayza F Al Fayez
BSc MSc PhD )
Assistant Professor
Ms. Areej Al-Turki
B.Sc.
Technician.
23-486
2/670
23-468
2/659 23-470
Zain Mohd.AL-Shareef 2/653 23-489
B.Sc.
Technician.
Hana Abdullah Basaffar 2/620 23-436
B.Sc.
Technician.
Rehab Aboobakar Al-
Aydoos
B.Sc.
Technician.
2/690 23-467
Nada Saleh Al-Saykhan 2/652 23-611
B.Sc.
Technician.
Reem Foad Ghazali
B.Sc.
Technician.
2/623 23-438 areej_t2001@yahoo.com pop_it4@yahoo.com nadnoon@hotmail.com reem_g77@hotmail.com
14

The following icons have been used to help you identify the various experiences you will be exposed to.
Learning objectives
Content of the lecture
Independent learning from textbooks
Independent learning from the CD-ROM.
Independent learning from the Internet
Home Work
Problem-Based Learning
15

Self- Assessment (the answer to self-assessment exercises will be discussed in tutorial sessions)
The main concepts
16

17

18

LECTURE# (1) :
DEPARTMENT: Clinical Biochemistry
Tutor: Dr. Dr.
TEACHING LOCATION: Auditorium
By the end of this lecture, you will be able to:
1. Identify the importance of studying clinical biochemistry in college of medicine
2. Compare between water and other solvents.
3. Recognize water properties which make the universal solvent.
4.Appreciate the role of buffer in human body homeostasis

Molecular structure of water

Non-covalent forces

Properties of water

Ionization of water

Buffers

Water is essential for life. It covers 2/3 of the earth's surface and every living thing is dependent upon it. The human body is comprised of over 70% water, and it is a major component of many bodily fluids including blood, urine, and saliva.
1. Required Texts And Resources: Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Reading Handouts will be distributed
3- Lectures and power point presentation will be published on department website: www.kaau.edu.sa/faculties/medicine/dcbcweb

19



You have the opportunity to watch the CD-ROM about Water & Chemical
Bonds. You can access the CD-ROM during your spare time.
1-
2-
3-
4-
5-
Biochemistry of water: http://library.thinkquest.org/28751/review/biochem/2.html
Water http://www.chm.bris.ac.uk/motm/water/water.htm
Water animation http://www.rkm.com.au/ANIMATIONS/animation-water-molecule.html
Hydrogen bonding animation www.elmhurst.edu/~chm/vchembook/163boilingpt.html
All about chemical bonding http://www.promotega.org/UGA06004/covalent_bonds.html
1List different types of chemical bonds. In a table form compare different types of bonds and their biological importance.
Clinical Question
The human body is greater than 80% water, but a relatively small loss of water content, such as that created by vigorous exercise or diarrhea, can have profound physiologic consequences. From a biochemical point of view, and from what you have learned in this course so far, why would small changes in water content alter biochemical processes?
I- MCQ:
Which of the following is NOT a "weak" interaction?
A. hydrogen bonds
B. van der Waals forces
C. disulfide bonds
D. ionic interactions
2- What is the maximum number of hydrogen bonds that one water molecule can have with neighboring water molecules?
A. 1
B. 2
20

C. 3
D. 4
3- The pH of a 10
-4
M solution of HCl is
A. 3
B. 10
C. 4
D. 4.5
III- True / False a.
Electrostatic interactions occur between atoms have the same charge b.
In water molecule, Oxygen is highly electrophilic
c.
Water molecules are bound together through Ionic bonds
d.
Buffers are made up of a mixture of a weak acid with its conjugate base or a weak base with its conjugate acid.
e.
pH + pOH = 10 For any substance dissolved in water
21

Lectures # (2-6) : Protein structure function relationship
DEPARTMENT: Clinical Biochemistry
Tutor: Dr.
Student Notes:
.
TEACHING LOCATION: Auditorium
By the end of this lecture, you will be able to:
1. Relate the structural properties of particular proteins such as hemoglobin and myoglobin to their functions in human health and disease.
2. Discuss the genetic bases of protein structure and how molecular defects lead to diseases such as sickle cell anemia.
3. Be able to recognize the structure of the commonly found amino acids in specific proteins and explain the their importance.

Hemoglobin:

Quaternary structure of hemoglobin, cooperative binding of oxygen, effect of hydrogen ion and carbon dioxide
(Bohr effect)

Functional significance of DPG, fetal hemoglobin, abnormal hemoglobin,
(sickle cell anemia).

Myoglobin:

Globular proteins, myoglobin configuration and conformation.

Oxygen binding site.

Microenvironment and reversible oxygen binding.
22

2
2
1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey P : 25 - 42
2. Reading Handouts will be distributed
3- Lectures and power point presentation will be published on department website: www.kaau.edu.sa/faculties/medicine/dcbcweb



You have the opportunity to watch the CD-
ROM about Hemoglobin. You can access the
CD-ROM during your spare time.
23

1- Haemoglobin http://www.ebi.ac.uk/interpro/potm/2005_10/Page1.htm
2- Haemoglobin http://www.iscid.org/encyclopedia/Haemoglobin
3- Haemoglobin animations http://www.umass.edu/microbio/chime/hemoglob/2frmcon t.ht]
4- Oxygen dissociation curve http://www.bio.davidson.edu/Courses/anphys/1999/Dicken s/Oxygendissociation.htm
5- Oxygen dissociation curve 2 http://www.ventworld.com/resources/oxydisso/dissoc.html
6- Haemoglobin dissociation curve 3 http://www.manbit.com/Hbdiss.htm
7- Carbon dioxide and Oxygen transport http://cal.man.ac.uk/student_projects/2001/MNQC7NDS/h omepage.htm
2In a table form compare between
Hemoglobin and Myoglobin including the difference in the :
Structure, Function, Oxygen binding and Oxygen dissociation curve.
Clinical Question
A 67 – year old man presented to the emergency department with one week history of angina and shortness of breath. He complained that his face and extremities had a "blue color" .
His medical history included chronic stable angina trated with isosorbide dinitrate and nitroglycerin. Blood obtained for analysis was chocolate – colored. What is the possible diagnosis
I- Short Questions:
24

a- What of the effects of the following on oxygen dissociation curve of Hemoglobin
1- 2,3 – BPG
2- CO
2
3- pH b- What is the molecular defect which leads to the following?
1- Sickle cell disease
2- Methemoglobinemia
3- Thalassemias
II- MCQ:
1- Hemoglobin is
A.
A tetramer of 4 myoglobin proteins.
B.
A tetramer of four globin chains and one heme prosthetic group.
C.
A dimer of subunits each with two distinct protein chains (alpha and beta).
D.
A dimer of subunits each with two myoglobin proteins.
2- Hydrophobic amino acid sequences in myoglobin are responsible for
A.
Covalent bonding to the heme prosthetic group
B.
The folding of the polypeptide chain
C.
The reversible binding of oxygen
D.
b and c above
3- Cooperative binding of oxygen by hemoglobin
A.
Is induced by hemoglobin
B.
Is a result of different affinities for oxygen by each subunit protein
C.
Is induced by oxygenation
D.
Is a result of interaction with myoglobin
III- True / False f.
Proteins often consist of multiple subunits so that they may have different functions under different conditions.
g.
The porphyrin prosthetic group is held into the interior of globin molecules by covalent bonds to specific amino acid residues h.
Myoglobin has a greater affinity for oxygen than hemoglobin. i.
Cooperative binding and
25

allosterism of hemoglobin allow oxygen to be unloaded at low partial pressures of oxygen in the tissues. j.
The Bohr effect is a description of the effect of pH on hemoglobin, oxygen bound more tightly at
low pH (in tissues) and less tightly
at higher pH values.
26

LECTURE # (7) : Introduction to the enzymes
TUTOR:
TEACHING LOCATION: Auditorium
.

Enzymes are important for medical students for diagnosis and treatment

Enzymes are typically large globular proteins that accelerate reactions by million folds

Substrate, product, apoenzyme, holoenzyme, cofactor, coenzyme,
27

active site and catalytic site, all are terms used in study of enzymes

There are many differences between enzymes and inorganic catalysts as reversibility and specificity of enzyme action

Naming of enzymes is either convenient for everyday use, or more complete systematic name divided into six major classes

Enzyme specificity are absolute, relative, stereochemical and group
Remember that all reactions in the body are mediated by enzymes, which are protein catalysts that increase the rate of reactions without being changed in the overall process. Enzymology is important as inborn errors of metabolism are due to deficiency of some enzymes. Also assay of particular enzymes can help in diagnosis of many diseases. The most striking characteristics of enzymes are their catalytic power and specificity which is of many types .
1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey , chapter 27 ( pp:365-367)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Chapter
18 (pp:217-218)
28




You have the opportunity to watch the CD-
ROM about enzymes. You can access the
CD-ROM during your spare time.
1- http://web.indstate.edu/thcme/mwkin g/enzyme-kinetics.html
2- http://www.stolaf.edu/people/qiannin i/fashanimat/enzymes
3- http:// www.reachoutmichigan.org/funexpe riments/quick/eric/enzymes
4- http://www.pubmedcentral.nih.gov/a rticlerender.fcgi?artid
5- http://www.ncbi.nlm.nih.ov/entrez/qu ery/fcgi?cmd
6- http://www.wiley.com/legacy/college/ boyer/0470003790
7- http:/www.siu.edu/departments/bioc hem/som_pbl/ms_ppt_anim.html
8- http://www.lewport.wnyric.org/wana maker/animations/Enzymes%20 activ ity.
** Enzymes are biological catalysts that accelerate the rate of reaction, by using the library and the internet try to explain how this occurs.
Clinical Case: A 52-year-old-man
29

presented at ER of a hospital with severe chest pain which had present for the past hour. He had a 2-year history of angina of efforts. How can you use enzymes in?: a.
Diagnosis of this case
b. Treatment of it
I- Short Questions:
1Enumerate enzyme specificity
2Differentiate between enzymes and inorganic catalysts
II- MCQ:
1.Enzymes are: a) Proteins b) Chloroplasts c) Genes d) Mitochondria
2. Enzymes are catalysts. They increase the rate of chemical reactions by: a) Raising the activation energy b) Temporarily increasing the temperature c) Covalently binding the substrate d) Lowering the activation energy
3. Enzymes are classified by the: a) Size of the enzyme b) Size of the substrate c) Type of reaction d) Rate of reaction
4. Shown below is a graph describing energy versus reaction coordinate for a catalyzed and uncatalyzed reaction. Fill in the blanks with the letter that corresponds to each stage of the graph.
30

_____ ES
_____ S (substrate)
_____ P (product)
_____ Uncatalyzed reaction
III- Complete the following:
Some enzymes require another chemical to function. These helper compounds are called
__________. If a cofactor is covalently bound to the enzyme, it is called a __________ group. An enzyme with its cofactor attached is called a
__________, while an enzyme minus its extra component is called an __________.
31

LECTURE # (8) : Mechanism of enzymatic catalysis
TUTOR:
TEACHING LOCATION: Auditorium
.
∆G and ∆G ‡

Two models have been proposed to explain how an enzyme binds its substrate; lock-and-key model and induced-fit model

Lock-and-key model assumes that an enzyme active site will accept a specific substrate

Induced-fit model recognizes that there is much flexibility in an enzyme’s substrate . Accordingly, an enzyme is able to conform to a substrate

Transition state is state of maximum energy through which the enzymatic reaction proceeds. It is not an intermediate compound.
32

 The differences in free energy (∆G) between the transition state and substrate is the free energy of activation (∆G‡)

Enzyme can enhance the rate of reaction by four processes: general/acid base catalysis, covalent catalysis, substrate strain and entropy effect
When correctly positioned and bound on the enzyme surface, the substrate may be “ strained
” toward the transition state. At this point the substrate has been “ set up
” for acid-base and/or covalent catalysis. Proper and the nearness of the substrate with respect to the catalytic groups
( proximity effect ) contribute to decrease in entropy and so enhance the rate of enzymatic reaction .
1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey , chapter 27 ( pp:365-367)
2.Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Chapter
18 (pp:217-218)



You have the opportunity to watch the CD-
ROM about enzymes. You can access the
CD-ROM during your spare time.
33

1- http://web.indstate.edu/thcme/mwking/e nzyme-kinetics.html
2- http://www.stolaf.edu/people/qiannini/fash animat/enzymes
3- http:// www.reachoutmichigan.org/funexperiment s/quick/eric/enzymes
4- http://www.pubmedcentral.nih.gov/articler ender.fcgi?artid
5- http://www.ncbi.nlm.nih.ov/entrez/query/ fcgi?cmd
6http://www.wiley.com/legacy/college/boy er/0470003790
7- http:/www.siu.edu/departments/biochem/s om_pbl/ms_ppt_anim.html
8- http://www.lewport.wnyric.org/wanamaker/ animations/Enzymes%20 activity
9- http://www.bio.winona.edu/berg/308/oldex am/308exam1.txt
** How does the enzyme bind its substrate?
By using the library and the internet try to answer this question.
I- Short Questions:
1. What is meant by transition state
2. Enzymes can enhance the rate
34

of reaction. Enumerate the processes by which this occurs.
II- MCQ:
1. The minimum amount of energy necessary for a molecule(s) to react is the: a) Activation energy b) Free energy c) Thermal energy d) Potential energy
2. The state produced when two or more molecules collide with just the right energy and just the right orientation so that a chemical reaction might occur is:
a) Catalytic state
b) Transition state
c) Activation state
d) Transient state
3. One of the following statement describing the mechanism of enzyme action is
INCORRECT: a) Many enzymes have flexible structures that allow them to enfold their substrate b) The substrate is often distorted when it enters an enzyme-substrate complex c) Amino acid side chains involved in the formation of the active site center are usually close together in the amino acid sequence of the enzyme protein d) Amino acid side chain near the active site center often have a role in the catalytic process
4.Which of the following statement about enzyme catalyzed reaction is NOT TRUE: a) Enzymes form complexes with their substrate b) Enzymes increase the activation energy for chemical reaction c) Many enzymes change shape slightly when substrate binds d) Reactions occur at the “active site” of enzymes, where a precise quaternary orientation of amino acids is an important feature of catalysis
III- Complete the following:
35

Enzymes carry out different chemical reactions in catalysis. Fill in the blanks with the name of the mechanism that matches with the example described in each line. Mechanism names to choose from are: covalent catalysis , acid-base catalysis , and metal ion catalysis.
__________ catalysis may involve Glu, Asp, His, Lys, or Arg residues.
__________ catalysis may involve Na
+
, K
+
or Mg
2+
,
Ca 2+ .
__________ catalysis may involve serine proteases.
36

LECTURE # (9) : Enzyme kinetics
TUTOR:
TEACHING LOCATION: Auditorium
.

The enzyme (E) combines with its substrate (S) to form an enzymesubstrate complex (ES).

The ES complex can dissociate again to form E + S, or can proceed chemically to form E and the product P.

The rate constants k 1 , k 2 and k 3 describe the rates associated with each step of the catalytic process.

The initial velocity (V o ) at low substrate concentration is directly proportional to [S], while at high substrate concentration the velocity tends towards a maximum value
(V max
) which is independent of [S].

Michaelis-Menten equation
37

describes a “hyperbolic curve” of the relationship between [S] and velocity of the reaction

Lineweaver-Burk plot (double reciprocal plot) gives a straight line if V o is measured at different substrate concentration

Not only substrate concentration , but also pH and temperature can affect enzymatic activity
Kinetics is a study of the rate of changes of substrates to products. Kinetics properties for many enzymes reveals that, the rate of catalysis V varies with [S] in a hyperbolic manner which is expressed in
Michaelis-Menten equation. Lineweaver-Burk plot gives a straight line of relationship between [S] and the rate of reaction..
1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey , chapter 27 ( pp:365-367)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Chapter
18 (pp:217-218)



You have the opportunity to watch the CD-
ROM about enzymes. You can access the
CD-ROM during your spare time.
1.
http://web.indstate.edu/thcme/mwking
/enzyme-kinetics
2.htmlhttp://www.stolaf.edu/people/qian nini/fashanimat/enzymes
38

3.http:// www.reachoutmichigan.org/funexperim ents/quick/eric/enzymes
4.http://www.pubmedcentral.nih.gov/art iclerender.fcgi?artid
5.http://www.ncbi.nlm.nih.ov/entrez/que ry/fcgi?cmd
6.
http://www.wiley.com/legacy/college/ boyer/0470003790
7.http:/www.siu.edu/departments/bioch em/som_pbl/ms_ppt_anim.html
8. http://www.lewport.wnyric.org/wanama ker/animations/Enzymes%20 activity.
By using the library and the internet try to illustrate the relationship between substrate concentration and reaction velocity.
I- Short Questions:
a) Would you think that all enzyme assays need the same pH? Why?
b) What is the meaning of optimum temperature?
II- MCQ:
1. Which of the following does not influence enzyme activity? a) pH b) Temperature c) Product degradation d) Substrate concentration
2. The Michaelis constant K m is: a) Numerically equal to ½ V max b) Dependent on the enzyme concentration
39

c) Independent on pH d) Numerically equal to the substrate concentration that gives half-maximal velocity
3. What effect does temperature have on enzymes? a) Boiling will denature them, as will being too cold b) Boiling will not harm them, but being too cold will denature them c) Boiling and cooling will both reduce the speed of their rate d) Boiling will denature them, but cooling will only slow down their work
4. The Michaelis-Menten equation is V o
=V max
[S]/(K m +[S]).
Fill in the blanks with the letters shown to correctly label each part of the graph
_____ Vmax
_____ [S]
_____ V o
_____ Point used to determine the Km
III- Complete the following:
Often the kinetic values of an enzyme are plotted using the Lineweaver-Burk equation:
1/v o
=Km/Vmax·1/[S] +1/Vmax
40

Enter "yes" or "no" to indicate if the equation and graphical terms match in each statement.
_____ Y-intercept and -1/Km
_____ X-intercept and 1/Vmax
_____ Slope and Km/Vmax
41

LECTURE # (10) : Enzyme inhibitors
TUTOR:
TEACHING LOCATION: Auditorium
.

There are two broad classes of enzyme inhibitors: reversible and irreversible

Reversible inhibitors interact with an enzyme via noncovalent association

Irreversible inhibitors interact with an enzyme via covalent association

Competitive inhibitors binds only to enzyme (E) and not to enzymesubstrate complex (ES)

Noncompetitive inhibitors binds either to (E) and/or to (ES)

Uncompetitive inhibitors binds only to (ES) and not to (E)

Enzyme inhibition may affect Vmax or Km or both

Many natural occurring and man-
42

made compounds are irreversible enzyme inhibitors

There are therapeutic application for enzyme inhibition
A competitive inhibitor prevents the substrate from binding to its enzyme, while noncompetitive or uncompetitive do not. Competitive inhibitor increases
K m without change V max.
Noncompetitive inhibitor
.
decreases V max without change in K m . But uncompetitive inhibitor decreases both V max and K m
1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey , chapter 27 ( pp:365-367)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Chapter
18 (pp:217-218)



You have the opportunity to watch the CD-
ROM about enzymes. You can access the
CD-ROM during your spare time.
1- http://web.indstate.edu/thcme/mwking/e nzyme-kinetics.html
2- http://www.stolaf.edu/people/qiannini/fa shanimat/enzymes
43

3- http:// www.reachoutmichigan.org/funexperime nts/quick/eric/enzymes
4- http://www.pubmedcentral.nih.gov/articl erender.fcgi?artid
5- http://www.ncbi.nlm.nih.ov/entrez/query/ fcgi?cmd
6- http://www.wiley.com/legacy/college/boy er/0470003790
7- http:/www.siu.edu/departments/biochem
/som_pbl/ms_ppt_anim.html
8- http://www.lewport.wnyric.org/wanamak er/animations/Enzymes%20 activity.
**How can the irreversible inhibitors bind the active site of the enzyme?
By using the library and the internet try to explain how this occurs.
Clinical Case: A young girl was brought to the pediatric clinic with infected wound on her knee. The mother was instructed to give the child penicillin. The child had improved after a week. a) What the possible mechanism of action of this antibiotic?
b) What is the target enzyme of penicillin?
IShort Questions:
44

1.
Classify enzyme inhibitors
2.
Tabulate the effect of competitive, noncompetitive and uncompetitive on V max and K m
II- MCQ:
1. Which type of reversible enzyme inhibitor binds to the free enzyme and the enzymesubstrate complex? a) Noncompetititive b) Competitive c) Uncompetitive d) None of the above
2. In competitive inhibition, one of the following statement is CORRECT: a) V max is increased b) The concentration of active enzyme molecule is unchanged c) The apparent K m is increased d) The apparent K m is decreased
3. Enzyme action can be influenced by the presence of inhibitors. Which of the following statements correctly matches the type of inhibitor with its effect on an enzyme . a) Irreversible and Renders the enzyme permanently inactive b) Competitive and Inhibitor binds only to
ES complex, only important when[S] high, Vmax lower, Km lower c) Noncompetitive and Can be overcome with high [S], Vmax unchanged, Km higher
d) Uncompetitive and Cannot be overcome with high[S], Vmax lower, but Km unchanged
4. In Lineweaver-Burk plot below: a) Mention the type of inhibition of enzymatic reaction b) Which one of the lines of the plot represents the enzymatic reaction without inhibition?
45

III- Complete the following:
Any molecule that acts directly on an enzyme to lower its catalytic rate is called ---------. Enzyme inhibition may be of --------types. Reversible inhibition can be overcome by-----but this is not possible for ----------- inhibition.
46

LECTURE # (11) : Enzyme regulation
TUTOR:
TEACHING LOCATION: Auditorium
.

There are key enzymes in a metabolic pathway which can be regulated and hence control the pathway

The activity of these enzymes can be regulated by:

Changing the amount of enzyme by enzyme induction, enzyme repression, and enzyme degradation

Changing the activity of enzyme by allosteric regulation, feedback control, covalent modification, and activation by cleavage

Compartmentation of pathways is also a way of enzyme regulation
47

Control of a pathway occurs through modulation of the activity of one or more key enzymes in the pathway . The rate-limiting enzyme and the committed step enzyme can be regulated by changing the amount (enzyme induction, repression, or degradation), or changing the activity (allosteric regulation, feedback inhibition, covalent modification, or enzyme cleavage) or compartmentation of the pathways.
1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey , chapter 27 ( pp:365-367)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Chapter
18 (pp:217-218)



You have the opportunity to watch the CD-
ROM about enzymes. You can access the
CD-ROM during your spare time.
1- http://web.indstate.edu/thcme/mwking/e nzyme-kinetics.html
2- http://www.stolaf.edu/people/qiannini/fa shanimat/enzymes
3- http:// www.reachoutmichigan.org/funexperime nts/quick/eric/enzymes
4- http://www.pubmedcentral.nih.gov/articl erender.fcgi?artid
48

5- http://www.ncbi.nlm.nih.ov/entrez/query/ fcgi?cmd
6- http://www.wiley.com/legacy/college/boy er/0470003790
7- http:/www.siu.edu/departments/biochem
/som_pbl/ms_ppt_anim.html
8- http://www.lewport.wnyric.org/wanamak er/animations/Enzymes%20activity .
9- en.wikipedia.org/wik/Enzymes
10.www.elmhurst.edu/~chm/vchembook/
573 regulate
** By using the library and the internet try to illustrate covalent modification of an enzyme (e.g. glycogen phosphorylase)
IIShort Questions:
1.
List some of the molecular mechanisms by which the catalytic activity of enzymes is controlled.
2.
Using a graph, illustrate the kinetic behavior of an allosteric enzyme
II- MCQ:
1. Allosteric enzymes are large, oligomeric proteins that have catalytic sites for binding substrates and regulatory sites that bind effectors. The separate oligomers influence one another; they work cooperatively. This is evidenced by the characteristic rate curves for allosteric enzymes which have: a) Michaelis-Menten kinetics b) Hyperbolic kinetics c) Sigmoidal kinetics d) Regulatory kinetics
49

2. Some enzymes are first synthesized in an inactive form. These zymogens must undergo proteolytic cleavage to produce the active enzyme. Which of the following statements are true of proteolytic cleavage? a) It is reversible b) It is irreversible c) It is random d) It occurs in the region of zymogen synthesis
3. Increased synthesis of an enzyme is known as: a) Ac a) Activation
b) Inhibition
c) Induction
d) Repression
4. In the graph below a) Which one of the plot explains the behavior of an allosteric enzyme? b) What is the meaning of cooperativity?
III- Complete the following:
The rate- limiting enzyme in a pathway is the enzyme with the-------Vmax, while the enzyme catalyzing the committed step is -----------.
50

LECTURE # (12) : Enzymes in clinical practice
TUTOR:
TEACHING LOCATION: Auditorium
.

Enzymes are used clinically in three principal ways:
- in diagnosis and prognosis of various disease
- as analytical reagents in the measurement of activity of other enzyme or non-enzyme substances
- as therapeutic agents

RNA enzymes (hammerhead ribozymes) and catalytic antibodies are recently discovered

Site-directed mutagenesis is used for design a new drug therapy
51

Isoenzymes are forms of an enzyme which are structurally different but have similar catalytic properties. Measurements of the isoenzymes of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and creatine kinase (CK) are of clinical value. Studies have shown that catalysis for biochemical reactions are not limited to naturally occurring proteins. Sitedirected mutagenesis (modification of amino acid sequence of known enzymes) is used for design new drug therapy.
1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey , chapter 27 ( pp:365-367)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Chapter
18 (pp:217-218)



You have the opportunity to watch the CD-
ROM about enzymes. You can access the
CD-ROM during your spare time.
1- http://web.indstate.edu/thcme/mwking/ enzyme-kinetics.html
2- http://www.stolaf.edu/people/qiannini/f
52

ashanimat/enzymes
3- http:// www.reachoutmichigan.org/funexperi ments/quick/eric/enzymes
4- http://www.pubmedcentral.nih.gov/arti clerender.fcgi?artid
5- http://www.ncbi.nlm.nih.ov/entrez/quer y/fcgi?cmd
6- http://www.wiley.com/legacy/college/b oyer/0470003790
7- http:/www.siu.edu/departments/bioche m/som_pbl/ms_ppt_anim.html
8- http://www.lewport.wnyric.org/wanama ker/animations/Enzymes%20activity .
9- www.dcnutrition.com/Miscellaneous/D etail
**It was believed that all enzymes were proteins till a recent discovery , by using the library and the internet try to explain this statement.
Clinical Case: A 52-year-old-man presented at ER of a hospital with severe chest pain which had present for the past hour. He had a
2-year history of angina of efforts. a) What specific tests would you request from the biochemistry laboratory?
b) How can use of an enzyme share in the treatment of this condition?
53

I- Short Questions:
1.
Enumerate five enzymes used in clinical diagnosis and mention the major diagnostic use for each
2.
What are the causes of presence of non-plasma specific enzymes?
II- MCQ:
1. In normal blood, the alkaline phosphatase activity is derived mainly from:
a) Bone and small intestine
b) Bone and liver
c) Small intestine and placenta
d) Bone and placenta
2. How many different isoenzymes of normal
LDH can be identified by electrophoresis at pH 8.6 a) Two b) Three c) Four d) Five
3. LDH assays are useful in diagnosing diseases of the: a) Heart b) Pancreas c) Liver d) a and c
54

Lectures # (
13- a ) : Membrane Structure and transport
DEPARTMENT: Clinical Biochemistry
Tutor: Dr.
TEACHING LOCATION: Auditorium
By the end of this topic the you will be able to:
1. Comprehend the fluid mosaic model of biologic membrane.
2. Understand how change in membrane composition changes its function.
Chemical compositions of membranes:

Separation of cells and intracellular organells into different chemical compartments by membranes.

Lipids of membranes.

Distribution of membrane lipids.

Membrane proteins.

Carbohydrates of membranes.
Molecular structure of membranes:

The fluid mosaic model of biologic membranes.

Asymmetry of membrane.

Membrane fluidity
55

1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey P :
2. Reading Handouts will be distributed
3- Lectures and power point presentation will be published on department website: www.kaau.edu.sa/faculties/medicine/dcbcweb



You have the opportunity to watch the CD-
ROM about Membrane structure. You can access the CD-ROM during your spare time.
1- Membrane structure http://cellbio.utmb.edu/cellbio/membrane.htm
2- Biological membrane structure http://www.pnas.org/cgi/content/abstract/66/3/615
3- Biological membrane structure http://www.pnas.org/cgi/content/abstract/66/3/615
4- Membrane Composition http://www.pnas.org/cgi/content/abstract/66/3/615
56

1- Draw a figure for cell membrane, mention the basic composition for the membrane and explain how membrane structure serves its function?
I- Short Questions: a- Which component (s) of membranes give it its fluid characteristics? b- Which part of a membrane helps it keep its shape (prevents deformation)? c- How are proteins arranged in a membrane? What is the difference between a transmembrane protein and a peripheral membrane protein? d- What feature in a membrane helps to prevent freezing? Be specific.
II- MCQ:
1- Triacylglycerols cannot form lipid bilayers because they
A.
Have hydrophobic tails
B.
Do not have polar heads
C.
Cannot associate with cholesterol
D.
Have polar heads
2- In a typical eukaryotic plasma membrane
A. Proteins can move in and out of the bilayer
B. Lipids can move and diffuse through the bilayer
C. Some lipids can rotate within the bilayer
D.
All of the above
3- The arrangement of lipid bilayers and other components is the basis for the currently widely accepted description which is called the
A. Lipid bilayer model
B. Mosaic model
C. Diffusion model
57

D. Fluid mosaic model
III- True / False
A.
According to the current model of cell membrane structure, the two layers of lipids in the bilayer are nearly identical
B.
Cholesterol accounts for 20% to 25% of the mass of lipids in a typical mammalian plasma membrane
C.
The distribution of peripheral membrane proteins is generally identical on both sides of a given membrane.
58

Lectures #(13 - b) : Membrane Transport
DEPARTMENT: Clinical Biochemistry
Tutor: Dr.
TEACHING LOCATION: Auditorium
Student Notes:
. to:
By the end of this topic you will be able
1. Explain how molecules move through membranes and the forms of energy needed to derive this process and to differentiate between active and passive transport.
2. Appreciate the functional role of membrane proteins as pumps, gates, channels and receptors .
Movement of molecules across
 membranes :



59

1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey P :
2. Reading Handouts will be distributed
3- Lectures and power point presentation will be published on department website: www.kaau.edu.sa/faculties/medicine/dcbcweb



You have the opportunity to watch the CD-
ROM about Membrane transport. You can access the CD-ROM during your spare time.
1-Membrane transport 1 http://www.emc.maricopa.edu/faculty/farabee/biobk/B ioBooktransp.html
2-Transport across cell membrane http://users.rcn.com/jkimball.ma.ultranet/BiologyPage s/D/Diffusion.html
3-Membrane transport mechanisms http://physioweb.med.uvm.edu/bodyfluids/membrane.
htm
4- Cell membrane and transport mechanisms http://staff.jccc.net/PDECELL/cells/transport.html
60

1- In a table form, describe Major mechanisms used to transfer material, mention the characteristics of each of them including the factor affecting and the need for energy.
I- Short Questions:
1- Give the definition for the following: a- Simple diffusion b- Facilitated diffusion c- Uniport, antiport, symport d- Active transport e- Endocytosis
II- MCQ:
1- Very large molecules (macromolecules) can be transported across membranes by :
A. pores or channels with very large
B.
C. openings through the center active transport proteins diffusion down a concentration gradient
D. endocytosis or exocytosis
2- Another name for facilitated diffusion is
A. Active transport
B. Transverse diffusion
C. Lateral diffusion
D. Passive transport
3Facilitated diffusion (passive transport) through a biological membrane is:
A. generally irreversible
B. driven by the ATP to ADP conversion
C. driven by a concentration gradient
D. endergonic
III- True / False a- Proteins that transport water across cell membranes are called aquaporins. b- Symport and antiport proteins must be active transport proteins.
61

c- Active transport involves the conversion of
ADP to ATP. d- Endocytosis is the process by which cells take up large molecules e- In adipocytes and muscle, glucose enters by facilitated diffusion
62

LECTURE # (14) : carbohydrate Chemistry
DEPARTMENT: Clin. Biochemistry UTOR: Dr.
63

Dr.
TEACHING LOCATION: Auditorium
By the end of this lecture, you will be able to:
1. Identify the wide range of functions of carbohydrates.
2. Classify carbohydrates.
3. Differentiate isomers, epimers and enantiomers.
4. Identify cyclization of manosaccharides to know anomers, mutarotation and glycosides.
5. Discuss some important chemical reactions of manosaccharides.

Introduction

Functions of carbohydrates

Classification:
- Monosaccharides
- Disaccharides
- Polysaccharides

Homopolysaccharides

Heterapolysaccharides
64

1. Required Texts And Resources: Lippincott Illustrated
Reviews, 3 rd edition, Champe & Harvey
2. Reading Handouts will be distributed
3- Lectures and power point presentation will be published on department website: www.kaau.edu.sa/faculties/medicine/dcbcweb



You have the opportunity to watch the CD-ROM about
Carbohydrate Chemistry. You can access the CD-ROM during your spare time.
1- Properties of Biomolecules http://www.phschool.com/science/biology_place/biocoach/bioprop
/intro.html
2- Animated Biomolecules http://www.umass.edu/microbio/rasmol/scripts.htm
3- Basic Biochemistry of Biomolecules: http://web.indstate.edu/thcme/mwking/biomolecules.html
1. Describe and compare the structures of cellulose and amylopectin. What is the general function of each of these polymers? How are their polymeric structures suited to their cellular functions? How does glycogen structure differ from amylopectin? How is this important to the role of glycogen.
1- Both glycogen and cellulose are polymers of glucose,
65

however glycogen forms an open, water-filled structure, while cellulose is linear and rigid. Compare the sructures of these molecules and explain how they suit their respective functions.
2Cellulose and starch are both polymers of glucose, yet most mammals can use starch, but not cellulose, as a source of fuel. Explain.
I- Short Questions:
ADescribe the following, explain the difference between each pair:
1. D versus L sugars (give an example of each)
2. Aldose versus ketose (give an example of each)
3. α and β anomers of D-glucose (give an example of each)
4. Glucose versus glucosamine.
B- Give one example for each of the following:
1Ketohexose
2- Aldopentose
3- Aldotriose
4- Heteropolysaccharide
5- Homopolysaccharide
II- MCQ:
1- The sugar residues of amylose are:
(A)Glucose units liked in β-1,4 linkages
(B)Glucose units liked in α l,4-linkages
(C ) Both galactose and fructose units liked in α l,4linkages
(D) fructose units liked in α l,4-linkages
2 . Which of the following contains ketone group?
(A) Glucose
(B)Mannose
(C) Ribulose
(D)Galactose
3. Hydrolysis of sucrose yields :
66

(A)Two moles of glucose
(B)Glucose and fructose
(C )Galactose and fructose
(D)Glucose and mannose
III- True / False
1.
The chemical formula of a monosaccharide is C
6
H
12
O
6
2.
Monosaccharides are classified according to the functional group into trioses, tetroses, pentoses, hexoses, ….etc.
3.
3. Carbohydrates are hydrates of carbon in which hydrogen and oxygen are present with the same ratio in water (2:1)
4.
Oligosaccharides contains more than 10 sugar units.
5.
Asymmetric carbon atom is the carbon atom attached to four different atoms or groups.
67

LECTURE # (15 - 16 ) : Introduction to Metabolism, Bioenergetics, and the
Role of ATP
DEPARTMENT: Clin. Biochemistry Student Notes: .
TUTOR:
TEACHING LOCATION: Auditorium
By the end of this lecture, you will be able to:
1. Compare and contrast catabolism and anabolism
2. Outline the general mechanisms for regulating metabolic pathways through enzymes.
3. Understand the role of ADP/ATP in connecting catabolic and anabolic reactions. How does the structure of these molecules allow them to mediate energy requiring reactions?
4. Compare the two physiological mechanisms for the net synthesis of ATP
5. Be able to determine the direction in which electrons would pass in an oxidation-reduction couple, given either ΔG’ or ΔE’.
6. Be familiar with the relative oxidation reduction potentials of
NAD, FAD (FMN), ubiquinone, cytochromes c and a, and oxygen.
7. Understand the importance of various mitochondrial compartments in cellular energy production.
8. Explain the relationship between electron flow and ATP production in the mitochondria
(chemiosmotic theory of Peter
68

Mitchell).
9. Describe the components of the electron transport chain, including the points where common inhibitors act.
10. Discuss the mechanism of
ATP production in the mitochondria.
11. Understand the concepts of respiratory control and coupling.

Metabolic Pathways
1- Definition, classification
(Anabolism &
Catabolism)
2- Catabolic phase
3- Low and high energy bonds – ATP – ADP cycle
4- Mechanism of collection of energy

Regulation of Metabolism
1- Signals from within the cells
2- Communication between cells
3- Intracellular messenger systems

Electron transport chain (ETC)
1- Components of ETC
2- Electron transport
3- Chemiosmotic hypothesis
4- ATP synthesis
5- Inhibition of ETC
6- Uncouplers
69

In order to survive, humans must meet two basic metabolic requirements: we must be able to synthesize everything our cells need that is not supplied by our diet, and we must be able to protect our internal environment. In order to meet these requirements, we metabolize our dietary components through four basic types of pathways: Fuel oxidative pathways, fuel storage and mobalization pathways, biosynthetic pathways, and detoxification or waste disposal pathways.
1- Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey P: 69 – 82 &
P: 89 - 94
2. Reading Handouts will be distributed
3- Lectures and power point presentation will be published on department website: www.kaau.edu.sa/faculties/medicine/ dcbcweb



You have the opportunity to watch the CD-
ROM about Bioenergetics and introduction to metabolism . You can access the CD-
ROM during your spare time.
1- Overview of metabolism http://www.elmhurst.edu/~chm/vchembook/5900verviewmet
.html
2- Introduction to metabolism (PowerPoint) http://www.rit.edu/~pac8612/Biochemistry/503(703)/ppt/Intr oduction_to_Metabolism.ppt
70

3- Electro Transport chain(ECT) http://www.dentistry.leeds.ac.uk/biochem/lecture/etran/etra n.htm
4- Electro Transport chain(ECT) http://vcell.ndsu.nodak.edu/animations/etc/movie.htm
5- ECT Animation http://www.sp.uconn.edu/~terry/images/anim/ETS_slo w.html
6- ATP Synthesis Animation http://www.sp.uconn.edu/~terry/images/anim/ATPmit o.html
7- ECT Movie http://vcell.ndsu.nodak.edu/animations/etc/movie.htm
1- Regulation of metabolism is based on need, from your studying outline the factors regulating metabolism and how they work according to body needs?
2- Summarize the composition of each of the respiratory chain complexes I, III, and IV. Diagram the pathway of electron transfer in the mitochondrial inner membrane from matrix NADH to oxygen, including the smaller electron carriers, coenzyme Q and cytochrome?
1If sodium ions rather than protons were pumped across the mitochondrial membrane, would there be any energy available to couple to ATP synthesis?
2- Clinical Question
The desire for a quick weightloss drug has led to a number of disasters. In the 1930s, dinitrophenol was explored as a possible weight-loss aid, but
71

it had a variety of severe side effects, including hyperthermia and death. Why was dinitrophenol a bad idea?
Explain how dinitrophenol induces hyperthermia
I- Short Questions:
1- Give the definition for the following:
a- Anabolic reaction
b- Catabolic reaction
c- Free Energy Change
d- Electron Transport Chain
2. Enumerate components of Electron
Transport Chain, How many ATP molecules will be produced from oxidation of One NADH+H + ,
FADH
2
respectively, in the chain?
3- How cAMP activates some metabolic reactions and inhibits others?
II- MCQ:
1. In the mitochondria NADH and QH2 are oxidized by ____________.
(a) carbon dioxide
(b) hydrogen peroxide
(c) ozone
(d) oxygen
2. The synthesis of one molecule of ATP from ADP requires _________ to be translocated across the inner mitochondrial membrane.
(a) one proton
72

(b) about two protons
(c) hundreds of protons
(d) 1 mole of protons
3. The degradation of which class of biochemicals does not significantly contribute to the release of energy to cells?
(a) nucleic acids
(b) Proteins
(c) Lipids
(d) carbohydrates
III- True / False a.
The biochemical reactions that degrade molecules, such as nutrients, are called anabolic reactions b.
Metabolic pathways generally have easily distinguished starting and stopping points c.
All metabolic reactions occur in the cytosol of cells d.
In mammals the enzyme complexes of oxidative phosphorylation are in the inner mitochondrial e.
Most of the free energy needed to drive ATP formation in the mitochondria is the result of an electrical contribution from a charge gradient across the inner mitochondrial membrane matrix
.
73

LECTURE # (17) :
TUTOR:
TEACHING LOCATION: Auditorium
.
By the end of this lecture you will be able to:
Define glycolysis, form a working definition knowing the substrates and products involved and any other key intermediates produced.
2Trace tissue location of glycolysis, particularly tissues or cells in the body where the pathway is most important.
3Locate the cell site of glycolysis, where in the cell it occurs
(cytosol).
4Describe the sequence of events, the overall reaction sequence and the number of stages and reactions: a.
Material Flow:: Trace the fate of labeled carbon or other elements through the pathway b.
Energy flow: Trace the production and consumption of ATP c.
Electron flow: trace the production and consumption of reducing power
74

5 Identify the Key steps, either those which form major control sites or those which are main " branch points"
6Describe connections to other pathways especially citric acid cycle .

Overview of Glycolysis

Coupled Reactions in Glycolysis

First Phase of Glycolysis

Second Phase of Glycolysis

Metabolic Fates of NADH and
Pyruvate

Anaerobic Pathways for Pyruvate

Energetic Elegance of Glycolysis

Other Substrates in Glycolysis
The glycolytic pathway is employed by all tissues for the breakdown of glucose to provide energy ( in the form of ATP) and intermediates for other metabolic pathways.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby


75


You have the opportunity to watch the CD-
ROM about glycolysis. You can access the CD-ROM during your spare time.
1. Introduction to Glycolysis: http://www.terravivida.com/vivida/glyintro/
2. Glycolysis Lecture: http://web.indstate.edu/thcme/mwking/glyc olysis.html
3. Glycolysis Animation: http://www.johnkyrk.com/glycolysis.html
4. Glycolysis Home: http://biotech.icmb.utexas.edu/glyco lysis/glycohome.html
3List the enzymes that convert glucose to glyceraldehyde 3-PO
4
. Add to your list the substrates, products, and cofactors for each of these enzymes. It might be handy to make up a table for this.
4List the enzymes that convert glyceraldehyde 3-PO4 to pyruvate.
Add to your list the substrates, products, and cofactors for each of these enzymes. It might be handy to make up another table for this.
2Clinical Question
Predict the effect of a GLUT4 knockout (in mice) on the levels
76

of blood glucose before and after a meal. Search the Internet and report whether your predictions were confirmed by experiment.
Of what use is a GLUT4 knockout mouse?
3Clinical Case
Ahmed entered the stadium for the final lap of his marathon race. He was well ahead of his competitors. In the last few minutes, he became confused. In the stadium, he started running around the track in the wrong direction and then collapsed.
What went wrong?
I- Short Questions:
1- Give the cellular location of the glycolytic pathway .
2- Where is NADH produced ?
3. Which step produces ATP? Which one of these is considered to be
'substrate level phosphorylation?
4- Name the enzyme that makes anaerobic glycolysis possible by using up the NADH that accumulates.
5 -Consider the ten steps of glycolysis, starting with glucose. What would be the effect on pyruvate concentration (increase, decrease or none) of increasing the concentration of the following?
Give a brief (one sentence) explanation for your answer.
77

a) ATP b) AMP c) citrate d) fructose-1,6-bisphosphate e) fructose-2,6-bisphosphate
II- MCQ:
1- Which of the following metabolites does not regulate glycolysis in liver cells?
A. glucose-6-phosphate
B. fructose-6-phosphate
C. fructose-1-phosphate
D. fructose-2,6-bisphosphate
2-During kinase reactions, the role of magnesium ions is to
A. be catalytic metals at the active sites of the enzymes.
B. interact with the hydroxyl groups of the various sugar molecules.
C. interact with the negative charges on phosphate groups.
D. provide a bridging atom between substrate and product, stabilizing the transition state.
3- Which of the following is not a substrate for hexokinase?
A. glucose
B. fructose
C. galactose
D. mannose
3- How many ATP molecules are produced from glucose during
78

anaerobic glycolysis?
A. 0
B. 1
C. 2
D. 3
III- True / False a.
Four molecules of ATP are consumed per glucose during the hexose stage of glycolysis b.
The reaction catalyzed by
PFK-1 is metabolically reversible c.
Mammals can convert pyruvate to either ethanol or lactate depending on the availability of oxygen d.
Glucose is normally transported into cells by an active protein transport since the concentration of glucose inside cells is normally higher than that in the blood. e.
Two molecules of ATP are consumed per glucose molecule during the hexose stage of glycolysis
79

LECTURE # (18) :
(TCA)
TUTOR:
TEACHING LOCATION: Auditorium
.
By the end of this lecture you will be able to:
Define TCA, form a working definition knowing the substrates and products involved and any other key intermediates produced.
2.
Trace tissue location of TCA, particularly tissues or cells in the body where the pathway is most important.
3.
Locate the cell site of TCA, where in the cell it occurs
(Mitochondria).
4.
Describe the sequence of events, the overall reaction sequence and the number of stages and reactions: aMaterial Flow:: Trace the fate of labeled carbon or other elements through the pathway bEnergy flow: Trace the production and consumption of ATP cElectron flow: trace the production and consumption of reducing
80

power
5.
Identify the Key steps, either those which form major control sites or those which are main " branch points"
6.
Describe connections to other pathways related to carbohydrate, lipid and amino acids metabolism
7.
Describe the amphibolic aspects of TCA and the role of its intermediate in various metabolic process.
8.
Diagram the shuttles for the transport of cytoplasmic reducing equivalents into the mitochondria

Overview of TCA

Reaction of the TCA cycle a- Oxidative decarboxylation of pyruvate b- The eight sequential reactions of TCA

Energy produced by the cycle

Regulation of the TCA cycle
The tricarboxylic acid cycle (TCA) is also called citric acid cycle or Kreb's cycle.
It is the major final common pathway of oxidation of carbohydrates, lipids and proteins since their oxidations yield Acetyl
Co A. It also plays a major role in lipogenesis,gluconeogenesis, transamination and deamination.
81

1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey P: 107 - 114
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby



You have the opportunity to watch the CD-
ROM about TCA. You can access the CD-
ROM during your spare time.
1- TCA animation http://www.science.smith.edu/departments/Biology/
Bio231/krebs.html
2-TCA animation http://www.wiley.com/legacy/college/boyer/04700037
90/animations/tca/tca.htm
3- TCA http://www.sigmaaldrich.com/Area_of_Interest/Life_
Science/Metabolomics/Key_Resources/Metabolic
_Pathways/TCA_Cycle.html
4- Step by step Kreb’s cycle http://www.terravivida.com/vivida/tcasteps/
5- Biochemistry animations http://www.wiley.com/college/fob/anim/ a. Diagram the
,
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beginning with pyruvate, giving the names of all enzyme substrates and products and names of enzymes (no abbreviations). Indicate where NAD+,
NADH, FAD, FADH2, GDP, Pi, GTP, coenzyme A, H2O, or CO2 are substrates or products of reactions. b. What is the fate of reducing equivalents of FADH2 generated in Krebs Cycle?
1Clinical Question
A patient diagnosed with thiamine deficiency exhibited fatigue and muscle cramps.
The muscle cramps have been related to an accumulation of metabolic acid. What is the metabolic product leads to this condition? Explain your answer.
I- Short Questions:
1- What is the intercellular location of
TCA?
2- What is the sources of Acetyl
CoA?
3- What are the products of TCA?
4- Describe the functions of 2 intermediates of TCA?
5- Summarize the functions of TCA.
II- MCQ:
1- The citric acid cycle oxidizes pyruvate and some of the pathway intermediates are starting materials for many biosynthetic
83

pathways. This means the citric acid cycle is a/an
______________.
A.
amplifying pathway
B.
strictly catabolic pathway
C.
anaerobic pathway
D.
amphibolic pathway
2- In eukaryotes the enzymes of the citric acid cycle are found in the
_________.
A. cytosol
B.
mitochondria
C.
nucleus
D.
endoplasmic reticulum
3- The enzyme pyruvate translocase is located
______________.
A. in the cytosol
B.in the inner mitochondrial membrane
C. in the mitochondrial matrix
D. in the endoplasmic reticulum
III- True / False
1The overall goal of the citric acid cycle is to oxidize pyruvate, form reduced coenzymes and produce ATP
2The citric acid cycle is an anaerobic pathway that occurs in the mitochondria of eukaryotes.
3ATP is consumed by the pyruvate dehydrogenase complex during the synthesis of acetyl CoA
4The citric acid cycle can be viewed as a multi-step catalyst simply because it returns to its original state after each round of reactions.
5Isocitrate is more easily
84

oxidized than citrate because it has a secondary alcohol group, whereas citrate's alcohol group is tertiary
85

LECTURE # (19) : Gluconeogenesis
TUTOR:
TEACHING LOCATION: Auditorium
.
Define Gluconeogenesis, form a working definition knowing the substrates and products involved and any other key intermediates produced.
2Trace tissue location of gluconeogenesis, particularly tissues or cells in the body where the pathway is most important.
3Locate the cell site of gluconeogenesis, where in the cell it occurs (cytosol)
Except for the carboxylation of pyruvate, which occurs in mitochondria.
4Describe the sequence of events, the overall reaction sequence and the number of stages and reactions: a.
Material Flow:: Trace the fate of labeled carbon or other elements through the pathway b.
Energy flow: Trace
86

the production and consumption of ATP c.
Electron flow: trace the production and consumption reducing power of
5Describe the sequence of events, the overall reaction sequence and the number of stages and reactions
6Identify the Key steps, either those which form major control sites or those which are main " branch points"
7Describe connections to other pathways, especially glycolysis and the reciprocal regulation of both pathway
8Identify the non carbohydrate sources of gluconeogenesis.

Overview of gluconeogenesis

Substrates for gluconeogenesis

Reactions unique to gluconeogenesis

Regulation of gluconeogenesis
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Gluconeogenesis is ' production of glucose from non – carbohydrate sources'
For a period of starvation of longer than about 12 hours. This pathway is very important for tissues which require a continuous supply of glucose as a metabolic fuel such as the brain, red blood cells, kidney medulla, lens and cornea of the eye, testes and exercising muscle.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey p: 115 - 122
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby



You have the opportunity to watch the CD-
ROM about gluconeogenessis. You can access the CD-ROM during your spare time.
1- Gluconeogenesis http://web.indstate.edu/thcme/mwking/gluconeogenesi s.html
2- Gluconeogenesis http://www.answers.com/topic/gluconeogenesis
3- Gluconeogenesis animation http://www.wiley.com/college/fob/quiz/quiz15/15-
22.html
4- Gluconeogenesis powerpoint presentation http://www.sb.fsu.edu/~chapman/Classes/Bch4054/Co ntent/Gluconeogenesis.pdf
88

1. a. Write out the two sequential reactions catalyzed by Pyruvate
Carboxylase, giving names and structures of reactants and products, including the active site prosthetic group. What is the nature and significance of the linkage of the prosthetic group to the Pyruvate
Carboxylase enzyme?
b. Describe and explain the significance of the effect of acetyl coenzyme A on the
Pyruvate Carboxylase enzyme.
1Why would it be disadvantageous to the organism to have Glycolysis and Gluconeogenesis operating simultaneously within a cell? Briefly describe one example of reciprocal regulation of
Glycolysis and
Gluconeogenesis, involving an allosteric regulator. For the example chosen, write out the reaction catalyzed by the enzyme in each pathway, and indicate the nature of the effect of the regulator (e.g., inhibition or activation).
89

I- Short Questions:
1- Give the cellular location of the gluconeogenesis pathway .
2- Enumerate the sources for gluconeogenesis ?
3. Define futile cycle, relate it to gluconeogenesis?
4- What are the irreversible reactions unique to gluconeogenesis?
5- How many ATP are consumed for synthesis of one molecule glucose from 2 pyruvates
II- MCQ:
1- Gluconeogenesis is
A. The formation of glycogen
B.The formation of starches
C.The formation of glucose from noncarbohydrates
D.The formation of glucose from other carbohydrates
2- Gluconeogenesis uses the same enzymatic reaction of glycolysis except for
A. Pyruvate kinase
B.4 irreversible reactions in glycolysis
C.3 irreversible reactions in glycolysis
D.2 irreversible reactions in glycolysis
3- Any compound that can be converted to _____can be a precursor for gluconeogenesis
.
A. Citrate
B.Pyruvate
C.Oxaloacetate
D.b and c
III- True / False
1Glucagon increases the transcription of the gene
90

2for PEP carboxykinase in gluconeogenesis, while insulin decreases it.
Fructose bisphosphate is
2,6a modulator that can stimulate glycolysis either or gluconeogenesis, depending on cellular glucose concentrations.
3At glucagon high concentrations gluconeogenesis will be favored over glycolysis.
4The brain normally uses both glucose and fatty acids as energy sources.
5Glycerol is one of the precursor gluconeogenesis. for
91

LECTURE # (20) :
TUTOR:
TEACHING LOCATION: Auditorium
.
1. Define the pentose phosphate pathwaw , form a working definition knowing the substrates and products involved and any other key intermediates produced.
1Trace tissue location of pentose phosphate pathwaw, particularly tissues or cells in the body where the pathway is most important.
2Locate the cell site of glycolysis, where in the cell it occurs
(cytosol).
3Describe the sequence of events, the overall reaction sequence and the number of stages and reactions: a.
Material Flow:: Trace the fate of labeled carbon or other elements through the pathway b.
Energy flow: Trace the production and consumption of ATP
92

c.
Electron flow: trace the production and consumption reducing power of
4Identify the Key steps, either those which form major control sites or those which are main " branch points"
5Describe connections to other pathways, with the emphasis on the role of NADPH in other biochemical pathways

Overview of pentose phosphate pathwaw

Irreversible oxidative reactions

Reversible nonoxidative reactions

Uses of NADPH

Glucose 6 – phosphate
Dehydrogenase Deficiency
The pentose phosphate pathway occurs in the cytosol of the cell. The pathway provides a major portion of body's
NADPH, which functions as a biochemical reductant. It also produces ribose 5- phosphate required for the biosynthesis of nucleotides.
93

1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey P: 143- 154
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby



You have the opportunity to watch the CD-
ROM about pentose phosphate pathway.
You can access the CD-ROM during your spare time.
1- Pentose phosphate pathway http://web.indstate.edu/thcme/mwking/pentosephosphate-pathway.html
2- Pentose phosphate pathway http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/m b2/part1/pentose.htm
3- Pentose phosphate pathway (PowerPoint) presentation www.cwu.edu/~geed/543/ Pentose %20 Phosphate %20 Pat hway .ppt
4- Pentose phosphate pathway (poweeroint ) www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/15pentose .ppt
1- Diagram and describe the mechanism of the reaction catalyzed by the enzyme
Transaldolase, including structures of major intermediates.
2. Write out the linear (oxidative) portion of the Pentose Phosphate
Pathway, giving names and structures of substrates and reactants, and the name of each enzyme. Summarize in words what happens at each step. What two
94

products of the linear portion of the
Pentose Phosphate Pathway have essential roles in anabolic metabolism? What are these roles?
Clinical Question
1- In male patients who are homozygous for glucose 6- phosphate dehydrogenase (G6PD) deficiency, pathophysiologic consequence are more apparent in erythrocytes (RBCs) than in other cells, such as in the liver. EXPLAIN.
I- Short Questions:
1- Give the cellular location of the pentose phosphate pathway
2- What are the main products of pentose phosphate pathway ?
3. Enumerate sources of NADPH?
4- What are the uses of NADPH in our body. Enumerate? .
5 - What are the effects of glucose 6- phosphate dehydrogenase deficiency
II- MCQ:
1- The major regulatory step of the pentose phosphate pathway is catalyzed by which enzyme?
A. transaldolase
B. phosphofructokinase-1
C. glucose 6-phosphate dehydrogenase
95

D. ribose 5-phosphate isomerase
2- The non-oxidative stage of the pentose phosphate pathway produces substances that are intermediates of ___________.
A. glycolysis
B. the citric acid cycle
C. the Cori cycle
D. glycogenolysis
3- Which is not a function of the main products of the pentose phosphate pathway?
A. To maintain the reduced form of iron in hemoglobin.
B.To provide reducing power for the synthesis of fatty acids.
C. To serve as precursors in the biosynthesis of RNA and DNA.
D.To raise the concentration of cAMP.
III- True / False
1Glucose 1,6-bisphosphate is the primary starting substrate for the pentose phosphate pathway.
2Two molecules of NADPH are generated for each molecule of glucose 6-phosphate that enters the pentose phosphate pathway.
3Rapidly dividing cells generally have a high pentose phosphate pathway activity.
4The enzyme transketolase transfers 2-carbon units from ketose phosphates to aldose phosphates
5NADH and pentose are the main products of pentose phosphate pathway
96

LECTURE # (21): Metabolism of Monosaccharides and Disaccharides
TUTOR:
TEACHING LOCATION: Auditorium
.
By the end of this lecture you will be able to:
Define pathways for fructose,
Galactose and Lactose
Metabolism , form a working definition knowing the substrates and products involved and any other key intermediates produced.
2Trace tissue location of these pathways, particularly tissues or cells in the body where the pathway is most important.
3Locate the cell site of these pathways, where in the cell it occurs (cytosol).
4Describe the sequence of events, the overall reaction sequence and the number of stages and reactions: a.
Material Flow:: Trace the fate of labeled carbon or other elements through the pathway b.
Energy flow: Trace
97

the production and consumption of ATP c.
Electron flow: trace the production and consumption of reducing powerIdentify the Key steps, either those which form major control sites or those which are main " branch points"
5Describe connections to other pathways especially Glycolysis

Overview

Fructose metabolism

Galactose metabolism

Lactose synthesis
Although many monosaccharides have been identified in nature, only a few sugars appear as metabolic intermediates or as structural components in mammals.
The major source of fructose is sucrose, while the major dietary source of galactose is lactose, entry of both to cells is insulin - independent both can be metabolized for energy generation through glycolysis.
98

1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey P: 135 - 142
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby



You have the opportunity to watch the CD-
ROM about Monosaccharide metabolism.
You can access the CD-ROM during your spare time.
1- Fructose Metabolism http://www.gpnotebook.co.uk/cache/1798635578.ht
m
2-Fructosa Web site http://www.drkaslow.com/html/fructose.html
3- Fructose and Galactose Metabolism http://web.indstate.edu/thcme/mwking/non-glucosesugar-metabolism.html
4- Galactose Metabolism http://www.gpnotebook.co.uk/cache/1899298874.htm
Describe, how different monosaccharides can be interconverted, Explain the benefits of this interconversion to body metabolism.
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1Clinical Question
A newborn baby experienced abdominal distension, severe bowel cramps, and diarrhea after being fed milk. A hydrogen analysis of his exhaled breath discovered an eighty – fold increase in the production of H
2
ninety minutes after milk feeding. aExplain the case b This condition is preventable or not?
2 Clinical Case
A galactosemic female is able to produce lactose, How?
I- Short Questions:
1- What is the main source of dietary fructose?
.
2- What is the main source of dietary galactose?.
3. Although fructose is more sweaty than Glucose it is not recommended to be an exchange to it in our diet
4- What is the effects Galactokinase deficiency.
5- Define fructosuria, what is its cause.
II- MCQ:
1- 1- Which is an intermediate formed in the conversion of glucose
100

to fructose?
A. glucose-1-phosphate
B. sorbitol
C. ribose
D. aldose reductase
2- What types of reactions are involved in the two-step conversion of glucose to fructose?
A. reduction followed by oxidation
B. two sequential hydrolysis reactions
C. hydrolysis followed by isomerization
D. phosphorylation followed by dephosphorylation
3- What is a cause of cataracts in the eye lens of individuals with diabetes?
A.Accumulation of sorbitol and protein precipitation in the lens.
B. Precipitation of glucose not oxidized by glycolysis in the lens.
C. The absence of membrane transport proteins for pyruvate in the lens cells.
D. Lack of regulation of gluconeogenesis in the lens and the accumulation of fructose.
III- True / False
1-
Lactose is the main source of dietary fructose.
2- Absorption of fructose is insulin dependent.
3- Aldose reductase reduces glucose, producing sorbitol
4- Classic galactosemia results from missing of Galactose kinase
5- Lactose is synthesized by lactase synthase from UDP glucose and
Galactose.
101

102

LECTURE # (22-23) : Structure of lipids
DEPARTMENT: Clin. Biochemistry Student Notes: .
103

TUTOR: Dr.
Dr.
TEACHING LOCATION: Auditorium
By the end of this lecture, you will be able to:
1. Identify the functions of lipids
2. Classify lipids.
3. Discuss the different basis for classification of fatty acids.
4. Identify the structure of different substances of compound lipids.
5. Discuss the structure and functions of different steroid compounds.
6. Identify caratenoids and their importance to human body.

Introduction

Functions of lipids

Fatty acids:
- Saturated
- Unsaturated

Simple lipids

Conjugated lipids:
- phospholipids
- glycolipids
- lipoproteins

Derived lipids
- steroids
- caratenoids
Lipids are substances insoluble in water and soluble in fat solvents. They are utilized in the body. They are classified into simple, conjugated and derived Lipids
104

1- Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Reading Handouts will be distributed
3- Lectures and power point presentation will be published on department website: www.kaau.edu.sa/faculties/medicine/dc bcweb



You have the opportunity to watch the CD-
ROM about Lipid Structure. You can access the CD-ROM during your spare time.
1- Properties of Biomolecules http://www.phschool.com/science/biology_pla ce/biocoach/bioprop/intro.html
2- Animated Biomolecules http://www.umass.edu/microbio/rasmol/script s.htm
3- Basic Biochemistry of
Biomolecules: http://web.indstate.edu/thcme/mwking/biomol ecules.html
1- In table form, describe different types of conjugated lipids, mention their biological importance
1- Both Lipids and Carbohydrates are organic compounds, formed mainly from Hydrogen, Oxygen and Carbon,
Explain why energy content of lipids is greater than carbohydrates.
105

I- Short Questions:
1- How can you tell whether a particular fatty acid is, or is not, essential?
2. How many fatty acids contain 18 carbon atoms, enumerate?
3- Enumerate different types of glycerophospholipids.
4- What is main differences between
HDL and LDL?
5- Give 2 example for hormones derived from cholesterol .
II- MCQ:
1. True statements about lipids include the following except:
(a) They are an intracellular energy source
(b) They are poorly soluble in water
(c) They are structural components of membranes
(d) They are composed of only carbon, hydrogen and oxygen
2. Fatty acids that are dietary essentials in humans include which of the following:
(e) Palmitic acid
(f) Stearic acid
(g) Oleic acid
(h) Linoleic
3. A choline residue is present in which of the following lipids?
(e) Phosphatidic acid
(f) Ganglioside
(g) Cholesterol
106

(h) Sphingomyelin
III- True / False
1Sphingosine is the backbone of lecithin
2Palmitic acid is an essential fatty acid
3Stearic acid is non essential fatty acid
4Chylomicrons are synthesized in the small intestine, formed mainly of triglyceride.
5Vitamin E is one of cholesterol derivatives
107

LECTURE # (24-25) : Fatty acid oxidation
TUTOR: .
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1- Sequence the reactions catalyzed by acyl-
CoA synthetase, carnitine palmitoyl transferaseI,carnitine-acylcarnitine translocase, and carnitine palmitoyl transferaseII.
2- Indicate which of these enzymes is regulated by malonyl-CoA.
3- Explain the importance of carnitine for the oxidation of long-chain fatty acid but not short- and medium-chain fatty acids.
4- Discuss the steps of beta oxidation of fatty acids.
5- Explain the importance of citric acid cycle for the oxidation of fatty acids .
6- Calculate the yield of ATP that is formed through the complete oxidation of one mole of palmitic acid.
7- Indicate the roles of long chain-, medium chain- and short chain-acyl CoA dehydrogenases in the oxidation of long chain fatty acids. Explain why a defect in the medium chain enzyme results in excretion of dicarboxylic acids and acyl carnitine esters in the urine.

Tissues that are most active in oxidation of fatty acids and its sub
108

cellular location

Conditions which favor the process of oxidation of fatty acids

Role of carnitine in the process of beta oxidation

The difference in oxidation of long, medium and short chain fatty acids

The steps of beta oxidation

Energy gained of beta oxidation

Regulation of beta oxidation

The other types of fatty acid oxidation

Diseases associated with genetic abnormality of fatty acid oxidation
Beta oxidation takes place in fed state in certain tissues as a source of energy, in starvation it is stimulated to provide energy for many tissues as liver (which becomes completely dependent on it as a source of energy.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. (187-193)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. (pp 169-
173)



You have the opportunity to watch the CD-
ROM about fatty acid oxidation. You can access the CD-ROM during your spare time.
109

1http://www.dentistry.leeds.ac.uk/biochem/t hcme/fatty-acid-oxidation.html
2http://www.biocarta.com/pathfiles/betaoxi dationPathway.asp
3http://ull.chemistry.uakron.edu/Pathways/ b_oxidation/index.html
4http://www.biocarta.com/pathfiles/boxnpP athway.asp
5http://www.biocarta.com/pathfiles/h_cptPathway.asp
6http://www.brookscole.com/chemistry_d/temp lates/student_resources/shared_resources/an imations/carnitine/carnitine1.html
7http://www.biocarta.com/pathfiles/oddnum berchainPathway.asp
8http://www.biocarta.com/pathfiles/polyunsatfa ttyacidPathway.asp
1- Try to explain the sequence of events that takes place in the mitochondria during beta oxidation of fatty acid
2- Write down how beta oxidation is regulated
1- Clinical Question: In diabetic patient type I, do you expect that beta oxidation of fatty acid to be increased, decreased or normal and why?
110

2- Clinical Case: A three month old baby brought to you by his mother, she was complaining that the baby seemed to be lethargic and does not grow well, and he was on breast fed only. What possible reason(s) may be the cause of this condition?
I- Short Questions: a) Name the different types of oxidation of fatty acid b) List the difference between fatty acid synthesis and beta oxidation regarding hydrogen carrier used and the site they occur
II- MCQ:
1- Two high energy bonds are lost during
β oxidation of long chain fatty acids in: a) Transport of acyl carnitine inside the mitochondria b) Formation of acyl CoA inside the mitochondria c) Formation of acyl CoA in the cytoplasm d) Release of acetyl CoA from acyl CoA by the action of thiolase enzyme e) Non of the above
2- All the following are intermediate in
β- oxidation except: a) FADH b) NADPH c) NADH d) Acetyl CoA e) All of the above
111

III- True / False a- The major type of fatty acid oxidation in most of he tissues is omega oxidation b- Beta oxidation takes place in all tissues c- Compared to carbohydrate oxidation of fatty acids generate smaller quantity of ATP
112

LECTURE # (26) : Fatty acid synthesis
TUTOR:
.
TEACHING LOCATION: Auditorium
1-
Identify the substrate for the building of fatty acid and where it is made. Explain how this substrate is transferred to the cytoplasm.
2- Describe the importance of glycolysis in the cytosol for fatty acid synthesis.
3- Discuss how the fatty acid synthase enzyme complex works.
4- Explain how stearic acid and oleic acid are synthesized from palmitic acid and in what regions of the cell they are synthesized.
5- Describe the different mechanisms of shortterm control of the formation of fatty acids.
6- Indicate the roles of insulin and glucagon in the regulation.
9- Describe long-term control (enzyme induction) of the formation of fat. Indicate the enzymes that are subject to long term control.
10-Explain what prevents the liver from oxidizing fatty acids at the same time it is synthesizing fatty acids.

Tissue and subcellular location for fatty acid synthesis

Transport of the building block for fatty acid synthesis from the mitochondria to the cytoplasm

The generation of the substrates for fatty acid synthesis in the cytoplasm

The arrangement and structure of
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fatty acid synthesase enzyme

Energy requirement for fatty acid synthesis

Regulation of fatty acid synthesis

Elongation and unsaturation of fatty acid
Fatty acid synthesis is an anabolic process, so it is favored during the fed state and inhibited during starvation through hormonal regulation which also activates fatty acid oxidation, so during starvation fatty acid synthesis is shut down and oxidation is turned on. Acetyl CoA is the substrate for fatty acid synthesis which is produced in the mitochondria and has to be transported to the cytoplasm in the form of citrate.
In the cytoplasm the other requirement for fatty acid synthesis are present (NADPH and fatty acid synthesase complex)
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. (pp 179-
186)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. (pp 179-
184)



You have the opportunity to watch the CD-
ROM about fatty acid synthesis You can access the CD-ROM during your spare time.
1- http://ull.chemistry.uakron.edu/Path
114

ways/FA_synthesis/index.html#
2- http://www.genome.ad.jp/kegg/pathway
/map/map00561.html
3- http://ull.chemistry.uakron.edu/Pathways/FA_ synthesis/index.html
4- http://ull.chemistry.uakron.edu/genobc/
Chapter_24/
5- http://www.uic.edu/depts/mcam/mcbc/l ect_2004/lecture_27.pdf
1- Outline and contrast the processes of fatty acid synthesis and oxidation
2- Write down the source(s) of each of the substrate for fatty acid synthesis
1- Clinical Case
Obesity, which will be covered in the tutorial.
I- Short Questions: a) Name the key regulatory enzyme in fatty acid synthesis b) Using a diagram drew how this enzyme is controled
II- MCQ:
1- Acetyl groups for fatty acid synthesis is derived mainly from: a) Only glucose b) Ketogenic amino acids
115

only c) Beta oxidation of fatty acid d) Glucose and amino acids e) Non of the above
2- Acetyl CoA for fatty acid synthesis is produced in the cytoplasm by the action of: a) Acetyl CoA synthetase b) Citrate lyase c) Pyruvate dehydrogenase d) Thiolase e) Non of the above
II- True / False a) The synthesis of fatty acids occurs in the cytoplasm of the cells b) The key regulatory enzyme in fatty acid synthesis is the fatty acid synthesase c) Insulin hormone inhibits fatty acid synthesis d) Citrate lyase enzyme has no role in the process of fatty acid synthesis
116

LECTURE # (27) : Ketone bodies metabolism
TUTOR:
.
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1- Define what is meant by ketogenesis and ketolysis
2- Enumerate the different ketone bodies
3- Evaluate the process of synthesis of ketone bodies
4- Explain the role of the liver in ketogenesis
5- Explain why ketone bodies synthesis is activated during fasting and starvation

Definition of ketogenesis and ketolysis

The central role of the liver in ketogenesis and its inability to utilize them

The steps of ketogenesis in the liver

The steps of ketolysis in extra hepatic tissues

Regulation of ketogenesis and ketolysis
Remember that ketone bodies are used as a major source of energy during starvation in most of the tissues even the brain to decrease the process of gluconeogenesis from muscle protein and spare the glucose produced from gluconeogenesis mainly to the brain and for the tissues which do not have mitochondria such as RBCs.
117

1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. (pp 193-
197)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. (pp 173-
176)



You have the opportunity to watch the CD-
ROM about fatty acid oxidation. You can access the CD-ROM during your spare time.
1- http://www.biocarta.com/pathfiles/ke tonebodiesPathway.asp
2- http://ull.chemistry.uakron.edu/genobc/Chapter
_24/
3http://ull.chemistry.uakron.edu/Pathway s/ketone_bodies/index.html
1- Draw the steps of ketogenesis indicating the steps during which
CoA-SH is regenerated
2- Drew a graph connecting both beta oxidation and ketogenesis
1- Clinical Question: a) In type I diabetes, ketone
118

bodies tend to increase more than type II, explain why?
2- Clinical Case: A type I diabetic patient delivered to you in a coma in the ER , his blood glucose level was 500 mg/dl (27.8 mmol/L), his breath had the acetone charachtristic odour. a) What other investigation(s) would you like to do? b) What type of coma is it? c) What is the biochemical bases of this coma?
I- Short Questions:
1- Explain why ketogenesis is essential for energy production in the liver during starvation
2- Explain why the liver can not utilize ketone bodies
II- MCQ:
1- Regarding ketogenesis and ketolysis: a) Ketogenesis takes place in the mitochondria of extrahepatic tissues while ketolysis in the cytoplasm of extrahepatic tissues b) Ketolysis takes place in the cytoplasm of extrahepatic tissues c) Ketogenesis and ketolysis takes place in liver mitochondria d) Ketogenesis takes place in liver mitochondria only e) All of the above
2- Regeneration of CoA-SH takes place during ketogenesis in the step (s) of: a) formation of acetoacetyl
CoA b) formation of beta hydroxybutyrate from
119

acetoacetate c) formation of hydroxymethylglutaeyl
(HMG) CoA d) Both (a) and (c) e) All of the above
III- True / False
1- Ketogenesis takes place in all the tissues
2- Ketone body oxidation is the major source of energy for most of the tissues in the absorptive state
3- Ketogenesis takes place in the cytoplasm of the liver only
120

LECTURE # (28) : Phospholipids metabolism
TUTOR:
.
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1- Discussthe structures of phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, and cardiolipin.
2Explain the different mechanisms for complexlipid synhesis
3Explain what is meant by a ceramide. Outline how ceramide is synthesized beginning with palmitoyl-CoA and serine. Show how a ceramide is converted into sphingomyelin.
4Describe the genetic abnormalities associated with complex lipid metabolism
5Evaluate the medical importance of phospholipid and sphingolipids in the body

Structure of the different phospholipids

Importance and function of phospholipids

Structure and different types of sphyngolipids

Nomenclature of sphingolipids

The importance of complex lipids
Remember that phospholipids and sphingolipids are very important compounds in the body. They play many important roles such as blood clotting, membrane structure
(almost half of membrane lipid is phospholipids), signal transuduction, and
121

nervous function.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. (199-216)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. (pp 321-
327)



You have the opportunity to watch the CD-
ROM about lipolysis. You can access the
CD-ROM during your spare time.
1- http://www.med.unibs.it/~marchesi/lipsynt h3.html
2- http://www.merck.com/mmhe/sec23/ch282
/ch282d.html
3http://www.dentistry.leeds.ac.uk/biochem/t hcme/lipid-synthesis.html#sphingolipids
4http://www.lipidlibrary.co.uk
5http://www.biocarta.com/pathfiles/sphingol ipidPathway.asp
6http://www.biocarta.com/pathfiles/h_ceram idePathway.asp
7http://www.biocarta.com/pathfiles/pcsynth esispathway.asp
8http://www.biocarta.com/pathfiles/h_ptdins
Pathway.asp
Search for the importance of sphingolipids as signal
122

transduction
1- Clinical Question:
Comment respiratory syndrome (RDS).
on distress
2- Clinical Case
Leukocyte samples isolated from the blood of a newborn infant are homogenized and incubated with ganglioside
GM2. Approximately 47% of the expected normal amount of N-acetylgalactosamine is liberated during the incubation period. What is most likely the diagnosis?
1-
I Short Questions:
II- MCQ:
a) These molecules contain almost exclusively stearic acid at carbon
1 and arachidonic acid at carbon 2 b) Phosphatidylinositol 4,5bisphosphate, PIP
2
) is involved in the transmission of signals for cell growth and differentiation c) The synthesis of
involves CDP-activated
1,2-diacylglycerol condensation with myo -inositol d) It is considered one of the sphingolipids
a) Are composed of a ceramide
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backbone with a wide variety of carbohydrate groups b) They have exactly the same structure of sphingomylines c) They never contain Nacetylneuraminic acid (NANA) d) One of its subtype ; Cerebrosides has more than one sugar group
III- True / False
1- Lecithin is important in blood clotting mechanism
2- Ceramide is one of the classes of glycerophospholipids
3- Phosphatidic acid is the precursor of all sphingolipids
124

LECTURE # (29) : Disposal of amino acid nitrogen
TUTOR:
.
TEACHING LOCATION: Auditorium
1.
Discuss the process of transamination and the characters of the transaminase enzymes.
2.
Explain how the cooperation by the enzymes alanine aminotransferase and glutamate dehydrogenase is accomplished
3.
Evaluate the reaction of glutamate dehydrogenase as an example of oxidative deamination, and the control of glutamate dehydrogenase.
4.
Correlate the reactions catalyzed by glutamate dehydrogenase, glutamine synthetase, glutaminase, and aspartate aminotransferase,
5.
Evaluate the clinical importance of transaminase enzymes
6.
Evaluate the important role of alanine and glutamine in transport of amino groups between different tissues and the liver
125


Definition of tranamination and deamination

Site of transamination and deamination

Importance of transamination as a tool for synthesis of non essential amino acids

Co-enzyme of tranasaminases

The importance of glutamate dehydrogenase in nitrogen metabolism and its regulation

The other types of deamination

The central role of glutamate in amino group transfer.

The clinical application of transaminases
Transamination and deamination are the process through which the cell transfer and get rid of nitrogen , some times for formation of non essential amino acid as in transamination and sometimes for urea formation as in deamination.
Transaminase enzymes have absolute requirement for PLP as coenzyme and are present in most of the tissues. Liver transaminases have great clinical application as a parameter for liver function tests.
1.Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. Chapter 19
(pp:247-250)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Chapter
18 (pp:218-222)
126




You have the opportunity to watch the CD-
ROM about this topic in the library.
1- http://ull.chemistry.uakron.edu/Path ways/nitrogen_pool/index.html
2- http://www.elmhurst.edu/~chm/vche mbook/632oxdeam.html
3- http://www.elmhurst.edu/~chm/vche mbook/631transam.html
4- http://drnelson.utmem.edu/aadeg.ht
ml
5- http://www.mesomorphosis.com/arti cles/mcdonald/protein-04.htm
6- http://www.humboldt.edu/~rap1/C43
8.S07/C438Notes/C438nLec38.htm
7- http://www.albany.edu/faculty/cs812/ bio366/L08_AminoAcidMetab1.pdf
8- http://web.indstate.edu/thcme/mwkin g/nitrogen-metabolism.html
9- http://seqcore.brcf.med.umich.edu/m cb500/aasyl/aametab.html
1- Clinical Question
2- Clinical Case
III- Short Questions: a) Enumerate the characters of transaminases b) The major deamination
127

pathways in the body is through -------------- enzyme
IV- MCQ:
1) For amino acids to be used in energy production, its amino group should be removed (deaminated).
Which of the following statmnt is correct: a) The most active enzyme in deamination is
AST which ispresent in almost all tissues b) L- amino acid oxidase is responsible for deamination of most o the amino cids c) D- amino acid oxidase is very active in human and is responsible for domination of most of the amino acids d) L-glutamate dehydrogenase is the major enzyme of deamination
2) In chronic liver diseases,
ALT&AST are elevated, these enzymes action include all the following except: a) Formation of non essential amino acids b) Libration of free ammonia c) Amino group of alanine and asparatic acid by their action end up in lutamate d) Help in balancing amino acid pool between
128

essential and non essential amino acids
3) The coenzyme of L- glutamate dehydrogenase is: a) NAD b) FAD c) CoASH d) Lipoic acid
4) Transaminase reaction: a) Is reversible b) Requires the presence of PLP as coenzyme c) Is irreversible reaction d) Both a & b
V- True / False a) One of the two pairs of reactants in transaminase reaction is almost glutamate and α-ketoglutarate b) ALT exists in the cytoplasm and mitochondria of the tissues c) L-glutamate dehydrogenase is stimulated by high concentration of ADP & GDP d) All amino acids can be transaminated e) Vitamin B6 acts as coenzyme in some of the transaminases reaction
129

LECTURE # (30) : Urea cycle and detoxification of ammonia
TUTOR:
.
TEACHING LOCATION: Auditorium
1.
Trace the source of the two nitrogen in the urea molecule
2.
Drew the steps of the urea cycle and its site in the tissue
3.
Explain the reaction catalyzed by the enzyme carbamoyl phosphate synthetase and its regulation
4.
Discuss how NH4+ and aspartate share in the formation of urea
5.
Explain why the formation of one molecule of urea requires the hydrolysis of four-high energy phosphate groups of
ATP.
6.
Explain how the ammonium ion produced in different tissues is transported to the liver for urea synthesis
7.
Discuss the different inborn errors in the urea cycle and their consequences
8.
Explain the cause of increasing orotic acid in urine when there is ornithin transcarbamoylase deficiency.
9.
Discuss the causes of acquired
130

hyperammonemia
10.
Discuss the importance of benzoic acid and phenylbutyrate therapy in patients with a defect in the urea cycle.

Overview of the urea cycle

Importance of urea cycle

Location of the cycle

Steps of urea cycle

Energy utilization of the cycle

Regulation of the cycle

The sources of nitrogen in the urea molecule

Connection between the urea cycle and TCA cycle

Genetic abnormality and its consequence

Acquired hyperammonemia

Clinical correlation
Ammonia is toxic compound produced in most of the tissues during amino acid metabolism; it is transported to the liver in the form of alanine and glutamine in order to be converted into non toxic form (urea). The liver is only tissue that converts ammonia into urea through the urea cycle.
Remember that urea synthesis is energy consuming process taking place in both he mitochondria and the cytoplasm of liver cell. The key regulatory enzyme is CPSI. Failure of conversion of ammonia into urea results in hyperammonemia which may congenital or acquired with its toxicity especially on the nervous system.
131

1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey pp (251-257)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Pp (222-
224)



You have the opportunity to watch the CD-
ROM about urea cycle. You can access the CD-ROM during your spare time.
1- http://www.albany.edu/faculty/cs812/bio36
6/L09_AminoAcidMetab2.pdf
2- http://www.wiley.com/college/fob/quiz/quiz20/
20-8.html
3- http://www.ureacycle.com/#
4- http://www.ureacycle.com/about_ureacycle.as
p
5- http://www.humboldt.edu/%7Erap1/C431.F01/
C431Notes/C431n14Dec.htm
6- http://web.indstate.edu/thcme/mwking/ureacy cledisorders.html
7- http://www.elmhurst.edu/~chm/vchembook/63
3ureacycle.html
8- http://www.nucdf.org/
9- http://www.gwu.edu/~mpb/urea.htm
10- http://rarediseasesnetwork.epi.usf.edu/ucdc/i ndex.htm
11- http://ull.chemistry.uakron.edu/Pathways/urea
_cycle/index.html
12- http://www.sickkids.ca/HowellLab/custom/rsc hArginine.asp
13- http://www.idph.state.il.us/HealthWellness/fs/ urea.htm
132

Try to trace the sources of nitrogen in urea molecule and connect the urea cycle to the TCA cycle.
3- Clinical Question: what are the causes of acquired hyperammonemia?
4- Clinical Case: A 45 year old male with long history of hepatitis C was brought to the ER for acute mental status changes. The family reported that he has been very disoriented and confused over the last few days and has been nauseated and vomited blood. a) What is most likely test you would like to do, and what do you except the result to be?
I- Short Questions: a) What is the key regulatory enzyme in the urea cycle b) What are the types of hyperammonemia
II- MCQ:
1- The major two amino acids which transport ammonia from peripheral tissues to the liver are: a) Alanine and aspartic acid b) Glutamine and alanine c) Glutamine and aspartic acid d) Non of the above
2- The total energy loss in urea
133

synthesis is: a) Two molecules of ATP b) Four molecules of ATP in the form of 5 high energy bonds c) Three molecules of
ATP in the form of 4 high energy bonds d) Non of the above
3- The molecule(s) that is (are) regenerated during urea synthesis is: a) Carbamoyl phosphate b) Arginine c) Ornithine d) All of the above
4- Orotic acid will mainly be increased in: a) Argenenimia b) Argininosuccinate aciduria c) Type I hyperammonemia d) Type II hyperammonemia
III- True / False
1. In CPSI reaction one molecule of
ATP is used
2. The all five reactions of urea synthesis occur in the mitochondria
3. The highest concentration of glutaminase enzyme is present in he liver
4. During acidosis, the liver synthesizes large amount of glutamine which is transported to the kidney
5. The carbons of fumarate in the urea cycle can return back to the cycle in the form of asprtate
134

LECTURES # (31): Individual amino acid catabolism (glycine metabolism)
DEPARTMENT: Clinical Biochemistry .
TUTOR:
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1.
Describe how glycine is converted into glucose precursor
2.
Discuss the importance of glycine in collagen synthesis
3.
Enumerate the different compounds that are derived from glycine
4.
Explain how glycine is converted into serine and the reverse pathway
5.
Discuss the inborn error of glycine metabolism

Overview of the fate for the different amino acids

Glycine amino acid and its role in synthesis of many compounds such as creatine, heme, collagen and bile salts.

The conversion of glycine into serine and the role of THF

The role of glycine in detoxification

Glutathione formation and its physiological role
 The role of γ-glutamyl cycle in amino
135

acid uptake by the tissues

The genetic abnormality that is associated with glycine metabolism
Remember that glycine is non essential amino acid, glucogenic and enters in the formation of many compounds in the body.
Glutathione which is formed from three amino acids including glycine is very important compound in the body as antioxidant and detoxifying agent. Glycine also enters in the formation of creatine in the muscle and acts as source of energy in the muscle (creatine phosphate).
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. Pp (259-
279)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Pp (225-
232)



You have the opportunity to watch the CD-
ROM about individual amino acid metabolism. You can access the CD-ROM during your spare time.
4http://web.indstate.edu/thcme/mwking/a mino-acid-metabolism.html
5http://web.indstate.edu/thcme/mwking/in born.html#amino
136

6http://www.genome.jp/kegg/pathway/ma p/map01150.html
7http://seqcore.brcf.med.umich.edu/mcb5
00/aasyl/aametab.html
8http://www.dentistry.leeds.ac.uk/bioche m/thcme/aminoacidmetabolism.pdf
9http://www.med.unibs.it/%7Emarchesi/a ametab.html
10http://www.rpi.edu/dept/bcbp/molbioche m/BiochSci/sbello/new_page_2.htm
11http://www.sigmaaldrich.com/img/assets/
4202/MetabolicPathways_updated_02_0
7.pdf
12http://www.sigmaaldrich.com/sigma/gen eral%20information/inborn_errors_char t.pdf
13http://ghr.nlm.nih.gov/condition=primar yhyperoxaluria
14http://www.emedicine.com/med/topic302
7.htm
15http://www.kidneyatlas.org/book2/adk2_
12.pdf
3- Glycine is an important amino acid, its importance is due to it enters in the formation of many molecules and compounds in our body, try to enumerate these compounds and discuss the role of glutathione.
3- Clinical Question:
I- Short Questions:
1- Enumerate the most common compounds that are formed from glycine
2- Using a graph drew the gamma glutamyl cycle
137

I- MCQ:
4- Primary hyperoxaluria is due to: a) Deficiency of tryptophan hydroxylae enzyme b) Argininosuccinate lyase enzyme c) Deficiency in the conversion of glyoxylic acid into formic acid due to defect in the oxidative decarboxylation of this compound d) Deficiency in the renal tubular reabsorption of glycine
5- Regarding creatine formation: a) It requires the cooperation of two organs, the liver and the kidney b) Three amino acids shares in its formation (glycine, arginine and methionine) c) Both (a) and (b) d) Non of the above
6- Glycine is considered glucogenic amino acid as it can be converted into: a) Succinyl CoA b) α-ketoglutarate c) propionyl CoA d) pyruvate
III True / False
1. Formation of creatine from guanido acetate is by tansmethylation reaction and the methyl donor is SAM
2. Three amino acids
(glycine, arginine and methionine) enter in the formation of creatine
3. Glycine combines with propionyl CoA for heme
138

formation
4. Glutathione is tripeptides
(γ-glutamyl-cysteinylglycine)
139

LECTURES # (32) : Individual amino acid catabolism (Phenylalanine and tyrosine)
DEPARTMENT: Clinical Biochemistry .
TUTOR:
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1.
Recognize the important compounds derived from phenylalanine
2.
Discuss how tyrosine is formed from phenylalanine and the importance of the enzyme of this transformation
3.
Explain the genetic defects in these amino acid metabolism and their consequences.

Phenylalanine as essential amino acid

Tyrosine as non essential amino acid

The formation of melanin from tyrosine

Formation of thyroid hormone

Formation of adrenaline and nor adrenaline

Genetic abnormalities associated with phenylalanine and tyrosine metabolism
140

Remember that phenylalanine is essential amino acid while tyrosine is not as it can be synthesized from tyrosine. Tyrosine is a precursor for many compounds in the body e.g. thyroid hormone, melanin pigments and adrenaline. Genetic defects in phenylalanine metabolism represents high percentage of the most common defects in amino acid metabolism and include phenylketonuria, alkaptonuria , tyrosinosis and albinism.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. Pp (259-
279)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Pp (225-
232)



You have the opportunity to watch the CD-
ROM about individual amino acid metabolism. You can access the CD-ROM during your spare time.
1http://web.indstate.edu/thcme/mwkin g/amino-acid-metabolism.html
2http://web.indstate.edu/thcme/mwkin g/inborn.html#amino
3http://www.genome.jp/kegg/pathway/ map/map01150.html
4http://seqcore.brcf.med.umich.edu/m cb500/aasyl/aametab.html
141

5http://dwb.unl.edu/Teacher/NSF/C11/
C11Links/www.bact.wisc.edu/microte xtbook/metabolism/RespProteins.htm
l
6http://en.wikipedia.org/wiki/List_of_a mino_acid_metabolism_disorders
7http://www.dentistry.leeds.ac.uk/bioc hem/thcme/aminoacidmetabolism.pdf
8http://www.med.unibs.it/%7Emarche si/aametab.html
9http://www.rpi.edu/dept/bcbp/molbio chem/BiochSci/sbello/new_page_2.ht
m
10http://www.sigmaaldrich.com/img/ass ets/4202/MetabolicPathways_updated
_02_07.pdf
11http://www.sigmaaldrich.com/sigma/ general%20information/inborn_erro rs_chart.pdf
12http://www.pnas.org/cgi/content/full/
96/6/3160
13http://www.pubmedcentral.nih.gov/ar ticlerender.fcgi?artid=1144976
14http://www.lowvision.org/albinism.ht
m
15http://ghr.nlm.nih.gov/condition%3D tyrosinemia
16http://www.ncbi.nlm.nih.gov/entrez/d ispomim.cgi?id=203500
Using the net try to find out the percentage of these genetic abnormalities in the world and in KSA
4- Clinical Question: what are the different types of albinism?
5- Clinical Case: During screening for inborn error of metabolism in KAU hospital, a baby was discovered positive for phenylpyruvate in urine. What is
142

your possible diagnosis? What further investigation would you like to do?
I- Short Questions:
1- Enumerate the most common inborn error in phenylalanine and tyrosine metabolism
2- How melanin is formed from tyrosine
II- MCQ:
1- Alkaptonuria is due to deficiency of: a) Phenylalanine hydroxylase enzyme b) Tyrosine hydroxylase enzyme c) Homogentisic acid oxidase d) Tyrosine α-ketoglutarate transaminase
2- All are true about phenylketonuria except: a) Tyrosine amino acid in this case becomes essential amino acid b) There is increased secretion of phenyllactate in urine c) Mental retardation is constant feature in untreated cases d) It is due to deficiency of tyrosine hydroxylase enzyme
3- In tyrosinemia: a) Phenylalanine hydroxylase is deficient b) Tyrosine α-ketoglutarate transaminase and (or) parahydroxyphenyl pyruvate oxidase is (are) deficient c) Tyrosinase enzyme is absent d) All of the above
III True / False
143

1. Glutathione is an inhibitor for the enzyme homogentisic acid oxidase
2. Melanin is directly formed from phenylalanine
3. In phenylketonuria, tyrosine becomes essential amino acids
144

LECTURES # (33) : Individual amino acid catabolism (Tryptophan, aspartic acid , glutamic acid, alanine and serine)
TUTOR:
.
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1.
Discuss the central role of glutamic acid in amino acid metabolism
2.
Explain the connection between glutamic acid and aspartic acid
3.
Show how these two amino acid are both non essential and glucogenic
4.
Discuss how these two amino acids act as neurotransmitter
5.
Show how melatonin and serotonin are made from tryptophan
6.
Trace how tryptophan in its catabolism is both glucogenic and ketogenic
7.
Discuss the genetic defect in
Hartnup disease
8.
Explain the role of alanine in nitrogen transport
145


Central role of gutamic acid in amino acid metabolism

Important compound derived from glutamic acid

Aspartic and glutamic acids as neurotransmitters

Tryptophan amino acid as a precursor of many important compounds such as melatonin and serotonin

Inborn error in tryptophan metabolism (Hartnup disease)

Alanin as a major transporter of nitrogen among tissues
Remember that tryptophan is essential amino acid used for synthesis of many biological compounds such as melatonin, serotonin and niacin. It is both glucognic and ketogenic. Glutamic acid and aspartic acid are both non essential glucogenic amino acids.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. Pp (259-
279)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Pp (225-
232)



You have the opportunity to watch the CD-
146

ROM about individual amino acid metabolism. You can access the CD-ROM during your spare time.
1http://web.indstate.edu/thcme/mwkin g/amino-acid-metabolism.html
2http://web.indstate.edu/thcme/mwkin g/inborn.html#amino
3http://www.genome.jp/kegg/pathway/ map/map01150.html
4http://seqcore.brcf.med.umich.edu/m cb500/aasyl/aametab.html
5http://dwb.unl.edu/Teacher/NSF/C11/
C11Links/www.bact.wisc.edu/microte xtbook/metabolism/RespProteins.htm
l
6http://en.wikipedia.org/wiki/List_of_a mino_acid_metabolism_disorders
7http://www.dentistry.leeds.ac.uk/bioc hem/thcme/aminoacidmetabolism.pdf
8http://www.med.unibs.it/%7Emarche si/aametab.html
9http://www.rpi.edu/dept/bcbp/molbio chem/BiochSci/sbello/new_page_2.ht
m
10http://www.sigmaaldrich.com/img/ass ets/4202/MetabolicPathways_updated
_02_07.pdf
11http://www.sigmaaldrich.com/sigma/ general%20information/inborn_erro rs_chart.pdf
12http://www.emedicine.com/derm/topi c713.htm
13http://www.medstudents.com.br/nefr o/nefro3.htm
14http://www.biology.arizona.edu/bioch emistry/problem_sets/aa/glutamate.ht
ml
15http://www.springboard4health.com/ notebook/proteins_glutamic.html
16http://www.chemie.fuberlin.de/chemistry/bio/aminoacid/as
147

p_en.html
17http://www.anyvitamins.com/aspartic
-acid-info.htm
Using a diagram try to drew the interconversion of glutamine, glutamic acid and
α-ketoglutarate showing tissue location, enzymes and coenzymes.
1- Clinical Question: what do you expect the level of glutamine in CSF sample to be in a case of comma due to liver cell failure?
2- Clinical Case:
I- Short Questions:
1- How GABA is formed and what is its role in the CNS
2- Why tryptophan is considered both glucogenic and ketogenic amino acid
II- MCQ:
1- Concerning 5hydroxytryptamine: a) It is produced from tryptophan by decraboxylation and then hydroxylation and acts as neurotransmitter b) It is produced from tryptophan by hydroxylation then decraboxylation and acts as neurotransmitter c) It is produced from tyrosine by the action of tyrosine hydroxylase
148

d) Non of the above
2- All are true about melatonin except: a) It is derived from serotonin in the pineal gland b) It is regulatory of gonadal function c) Stimulates synthesis of GABA d) It has sleeping inducing effect
3- Concerning glutamic acid: a) It is essential amino acid b) It is ketogenic amino acid c) Shares in the formation of glutathione d) It is inhibitory neurotransmitter
1-
III True / False
1. Aspartic acid is non essential ketogenic amino acid
2. Tryptophan is the precursor of melanin
3. GABA is derived from glutamic acid and acts as inhibitory neurotransmitter
4. Aspartic acid enters in the formation of both purine and pyrimidine
149

LECTURES # (34 - a) : Individual amino acid catabolism (Valine, leucine and isoleucine)
Student Notes:
DEPARTMENT: Clinical Biochemistry .
TUTOR:
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1.
Describe the common pathway of catabolism for these three amino acids
2.
Trace the pathway to find out which of them is glucogenic or ketogenic or both
3.
Discuss the importance of these amino acids in muscle metabolism
4.
Explain the genetic defect associated with these amino acids

The common pathway for catabolism of these amino acids

The end product of those amino acids (glucogenic, ketogenic or both)

Enzymes necessary for their catabolic pathway

Enzymes defect in their catabolic pathway (Maple Syrup urine disease)
150

Remember that these amino acids have a common catabolic pathway. Their catabolic pathway after transamination has similar features to that of beta oxidation of fatty acids.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. Pp (259-
279)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Pp (225-
232)



You have the opportunity to watch the CD-
ROM about individual amino acid metabolism. You can access the CD-ROM during your spare time.
1http://web.indstate.edu/thcme/mw king/amino-acid-metabolism.html
2http://web.indstate.edu/thcme/mw king/inborn.html#amino
3http://www.genome.jp/kegg/pathw ay/map/map01150.html
4http://seqcore.brcf.med.umich.edu
/mcb500/aasyl/aametab.html
5http://dwb.unl.edu/Teacher/NSF/
C11/C11Links/www.bact.wisc.edu
/microtextbook/metabolism/RespP roteins.html
151

6http://en.wikipedia.org/wiki/List_ of_amino_acid_metabolism_disor ders
7http://www.dentistry.leeds.ac.uk/b iochem/thcme/aminoacidmetabolis m.pdf
8http://www.med.unibs.it/%7Emar chesi/aametab.html
9http://www.rpi.edu/dept/bcbp/mol biochem/BiochSci/sbello/new_pag e_2.htm
10http://www.sigmaaldrich.com/img
/assets/4202/MetabolicPathways_u pdated_02_07.pdf
11http://www.sigmaaldrich.com/sig ma/general%20information/inbor n_errors_chart.pdf
12http://www.emedicine.com/derm/t opic713.htm
13http://www.medstudents.com.br/n efro/nefro3.htm
14http://www.biology.arizona.edu/bi ochemistry/problem_sets/aa/gluta mate.html
15http://www.springboard4health.co
m/notebook/proteins_glutamic.ht
ml
16http://www.chemie.fuberlin.de/chemistry/bio/aminoacid
/asp_en.html
17http://www.anyvitamins.com/aspa rtic-acid-info.htm
1- Clinical Question: Four types of MSD are present, on what basis this classification depends?
2- Clinical Case:
I-Short Questions:
152

a) What are the end product of catabolism of these branched chain amino acids b) What is the enzymatic defect in
MSU disease
II- MCQ:
1- All the following are branched chain amino acids except: a) Methionine b) Valine c) Leucine d) Isoleucine
2- The defect in MSU disease is due to deficiency of : a) Methionine synhesase b) Dihydro biopterine reductase c) Branched chain α-amino acid transferases d) Branched chain α- keto acid dhydrogenase complex
III True / False
1. Leucine is purely ketogenic amino acid
2. Branched chain amino acids are mainly
3. metabolized by the liver
The oxidative decraboxylation in the pathway of catabolism of branched chain amino acids requires the same coenzymes as those in the pyruvate dehydrogenase complex.
153

LECTURES # (34 - b) : Individual amino acid catabolism (sulfur containing amino acids)
TUTOR:
.
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1.
Discuss the function of both cystein and cystin in protein structure
2.
Explain how the SH groups in these amino acids are important in metabolism
3.
Show how SAM is formed and its importance
4.
Discuss the different inborn error associated with these amino acids

The catabolic pathway for cystein and cystin

Importance of cystein and cystin in protein structure

Inborn error of metabolism associated with these two amino acids

Formation of SAM and its importance as methyl donor

Relation of SAM metabolic pathway to cardiovascular disease
Remember that sulfur containing amino acids are important in protein structure
154

because of the SH groups, also this group is important in their function in detoxification. SAM is derived from methionine amino acid and it acts as a major methyl donor in the body in the transmethylation reactions.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey. Pp (259-
279)
2. Supplementary Texts And Resources:
Medical Biochemistry, Baynes and
Dominiczek, 1 st edition, Mosby. Pp (225-
232)



You have the opportunity to watch the CD-
ROM about individual amino acid metabolism. You can access the CD-ROM during your spare time.
1http://jn.nutrition.org/cgi/conte nt/abstract/136/6/1750S
2http://en.wikipedia.org/wiki/H omocystinuria
3http://web.indstate.edu/thcme/ mwking/amino-acidmetabolism.html
4http://web.indstate.edu/thcme/ mwking/inborn.html#amino
5http://www.genome.jp/kegg/pa thway/map/map01150.html
6http://seqcore.brcf.med.umich.
edu/mcb500/aasyl/aametab.ht
ml
7http://jn.nutrition.org/cgi/conte
155

nt/abstract/137/2/339
Clinical Question: Try to explain the role of homocystein in cardiovascular diseases.
Clinical Case:
I-Short Questions:
II- MCQ:
1- Methionine adenosyl transferase a) Is the enzyme that regenerates methionine from homocysteine b) It is the enzyme that is responsible for SAM formation c) Deficiency of this enzyme will lead to homocysteinuria d) Non of the above
2- The major disposal pathway (s) of homocystein is (are): a) Conversion to methionine b) Formation of cysteine c) Both (a) and (b) d) Non of the above
III-
III True / False
E. During SAM synthesis ATP molecule is converted into
AMP
F. Homocysteinuria is due to deficiency in cystathionine synthase enzyme
G. Cystein amino acids is considered essential amino acid as it can not be synthesized in the body
156

LECTURE # 35 : Nucloetides Structure and Functions
DEPARTMENT: Clinical Biochemistry .
TUTOR:
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1.
List the different functions of nucleotides.
2.
Describe the general structure of nucleotides.
3.
Identify the structural components of nucleotides.
4.
Name different types of nucleotides.
5.
List different types of free
6.
nucleotides.
Discuss digestion absorption of nucleic acids. and

Importance of nucleotides.

General structure of nucleotides.

Nitrogenous bases.

Structure of nucleosides.

Structure of nucleotides.

Free nucleosides and nucleotides.

Digestion and absorption of nucleic acids.
Nucleotides are the basic building blocks of nucleic acids (DNA & RNA). In addition,
157

nucleotides play a very important role as the energy currency in cell metabolism. Most coenzymes are free nucleotides that play a major role in metabolism. Moreover, many nucleotides are present in our soft drinks along with tea, coffee and cocao.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Supplementary Texts And Resources:
Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about Structure of Nucleotides.
You can access the CD-ROM during your spare time.
1.
http://www.biochem.med.umich.edu/b iochem/courses/bc452/downloads/nucl eotides/nucleotide_metab_1.ppt
2. www.rpi.edu/~bellos/ Nucleotide s_revised.
ppt
3. http://www.ucd.ie/indmicro/patrick2/BL ec1(2004).ppt
4.
http://www.pharmacy.umn.edu/img/as sets/12622/lectures1and2.ppt
5. http://academics.vmi.edu/biochem/28-
Biosynthesis%20&%20Degradation%20 of%20Nucleotides.ppt
158

4Clinical Question
5Clinical Case
I. Short Questions:
1. What are the important nucleotide functions?
2. Identify the components of nucleosides and nucleotides.
3. List Five of the important free nucleotides:
II. MCQs:
1. A nucleoside is composed of:
a. Base and ribose sugar.
b. Base, ribose sugar and phosphates.
c. Base and phosphates.
d. Ribose and phosphates.
2.
The following nucleotide acts as a second messenger:
a. AMP.
b. cAMP.
c. SAM.
d. NADH.
3 . The following nucleotide acts as a methyl group donor:
a. AMP.
b. cAMP.
c. SAM.
d. NADH.
III. True / False
1. cAMP acts as a coenzyme for
159

dehydrogenase enzymes.
2. A nucleotide consists of a nitrogenous base, a pentose sugar
and phosphate group.
3. ATP consists of adenine, deoxy ribose and triphosphate
group.
4. NAD + and NADP + act as coenzymes for dehydrogenase
enzymes.
5. Digestion of nucleic acids occurs mainly in the stomach.
160

LECTURE # 36 - a: Purine Nucleotide Metabolism
TUTOR: .
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1- List the different sources of atoms of purine ring.
2- Define de novo pathway and salvage pathway
3. List the main stages of de novo synthesis of purine.
4- State the rate limiting step of de novo synthesis of purine.
5. Describe synthesis of IMP.
6. Describe conversion of IMP to
AMP & GMP.
7. Outline regulation of
Biosynthesis of Purine Nucleotides
8. Outline regulation of
Interconversion
Nucleotides of Purine
9. List synthetic inhibitors of de novo purine synthesis and their sites of effect.
161

10. Describe salvage pathway of purine synthesis.

De novo synthesis of purine nucleotides.

Regulation of Purine Synthesis.

Salvage pathway of purine synthesis.
In purne synthesis, the purine ring atoms are added directly on the ribose phosphate giving rise to the IMP nucleotide. IMP then acts as the start point of synthesis of both AMP and
GMP nucleotides.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about Purine nucleotide metabolism You can access the CD-
ROM during your spare time.
1.
http://www.biochem.med.umich.edu/b iochem/courses/bc452/downloads/nucl eotides/nucleotide_metab_1.ppt
2.
162

www.rpi.edu/~bellos/ Nucleotide s_revised.
ppt
3. http://www.ucd.ie/indmicro/patrick2/BL ec1(2004).ppt
4.
http://www.pharmacy.umn.edu/img/as sets/12622/lectures1and2.ppt
1. http://academics.vmi.edu/biochem/2
2.
8-
Biosynthesis%20&%20Degradation
%20of%20Nucleotides.ppt
6- Clinical Question
7- Clinical Case
I. Short Questions:
1. Contrast and compare the de novo synthesis, salvage pathways
in nucleotide synthesis.
2. What is the significant nucleotide role of inosine monophosphate
(IMP)?
3. How are ATP and GTP derived?
II. MCQs:
1. The amino acids involved in purine synthesis include all the
following EXCEPT:
a. Aspartic acid.
b. Glutamine.
c. Glycine.
d. Glutamic acid.
163

2. The committed step in purine biosynthesis is:
a. Synthesis of carbamayl phosphate.
b. Synthesis of orotic acid.
c. Synthesis of phosphoribosylamine.
d. Synthesis of IMP.
3. The first nucleotide formed in purine biosynthesis is:
a. AMP.
b. GMP.
c. IMP.
d. ATP.
III. True / False:
1.
De novo synthesis of nucleotides is their synthesis from preformed bases resulting from cell turn over or from diet.
2.
Salvage of nucleotides is their synthesis from low molecular weight precursors.
3.
HMP provides ribose-5- phosphate for synthesis of nucleotides.
4.
The rate limiting step of purine synthesis is catalyzed by PRPP synthase.
5. Purine nucleotides are synthesized by addition of different
atoms to the NH2 group of phosphoribosylamine.
164

165

LECTURE # 36- b : Purine Nucleotide Metabolism
TUTOR:
TEACHING LOCATION: Auditorium
.
By the end of this lecture you will be able to:
1. Discus catabolism of Purine
Nucleotides and identify key enzymes.
2. List factors affecting plasma urate
3. List causes of increased plasma urate
4. Define hyperuricemia.
5. State metabolic classification of gout.
6. State clinical classification of gout.
7. Discus the biochemical basis of treatment of gout.
8. Discus the biochemical basis of treatment of Lesch-Nyhan syndrome.
9. Discus the biochemical basis of treatment of SCIDS.
10. Define hypouricemia and its causes .
166


Catabolism of Purine Nucleotides.

Clinical Significances of Purine
Metabolism:
- Uric acid.
- Factors affecting plasma urate
- Causes of increased plasma urate
- Clinical considerations of Purine
Catabolism:
1. Gout.
2. Lesch-Nyhan syndrome.
3. Severe combined immunodeficiency syndrome (SCIDS).
4. Hypouricemia.
The defect in purine metabolism is due to accumulation of uric acid, the end product of purine catbolism. This may result from a defective enzyme in the purine metabolism or as a consequence of other diseases. The problem with uric acid accumulation is due to its limited solubility and its precipitation as crystals in the soft tissues. This promotes an immune response with the consequent symptoms. The proper treatment involves blocking the enzyme xanthine oxidase with allopurinol a competitive inhibitor.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
167

Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss


You have the opportunity to watch the CD-
ROM about Purine nucleotide metabolism.
You can access the CD-ROM during your spare time.
1- http://www.biochem.med.umich.edu/bioc hem/courses/bc452/downloads/nucleotid es/nucleotide_metab_1.ppt
2- www.rpi.edu/~bellos/ Nucleotide s_revise d.
ppt
3- http://www.ucd.ie/indmicro/patrick2/B
Lec1(2004).ppt
4- http://www.pharmacy.umn.edu/img/as sets/12622/lectures1and2.ppt
5- http://www.ninds.nih.gov/disorders/le sch_nyhan/lesch_nyhan.htm
6- http://ghr.nlm.nih.gov/condition=lesch nyhansyndrome
7- http://www.nlm.nih.gov/medlineplus/e ncy/article/001655.htm
8- http://www.healthatoz.com/healthatoz/
Atoz/common/standard/transform.jsp
?requestURI=/healthatoz/Atoz/ency/le sch-nyhan_syndrome.jsp
9- http://www.niams.nih.gov/hi/topics/go
10. ut/gout.htm
www.medicinenet.com/ gout /article.htm
11. http://www.footphysicians.com/footankle info/gout.htm
12.
www.uclasfvp.org/rheum/10%20min%
20Rheum%20talks/Crystal%20arthritis/ G out .
ppt
13.
168

http://academics.vmi.edu/biochem/28-
Biosynthesis%20&%20Degradation%2
0of%20Nucleotides.ppt
Clinical Question:
Clinical Case:
I. Short Questions:
1. Outline the rate-limiting step for purine synthesis and its regulation.
2. Outline the conversion of IMP to purine nucleotide monophosphates and its
regulation.
3. Outline regulation of biosynthesis of purine nucleotides.
4. Outline regulation of interconversion of purine nucleotides.
5. What is the role of uric acid in nucleotide metabolism?
6. List the causes of increased plasma urate
7. Contrast and compare gout and Lesch-
Nyhan syndrome with regards to their
biochemical abnormalities.
II. MCQs:
1. Allopurinol is the drug of choice for treatment of: a. Gout.
b. Orotic aciduria.
c. Chronic renal failure.
d. Non of the above.
2. Primary gout:
a. Is characterized by accumulation and urinary excretion
169

of orotic acid.
b. May develop in the course of other disease.
c. Resulted from defect of the enzyme orotate phosphoribosyl
transferase.
d. Allopurinol is used in the treatment of this disease.
3. Which one of the following is NOT a cause of gout:
a. Leukemia and related malignancies.
b. Genetic diseases that limit the amount of phosphate available
for purine salvage reactions.
c. Decreased sensitivity of the regulated step in purine
biosynthesis to
its inhibitors.
d. Genetic defects that cause overproduction of pyrimidines.
4. The end product of purine metabolism in a person taking
allopurinol for gout is:
a. Orotic acid.
b. Hypoxanthine and Xanthine.
c. Urate.
d. dTMP.
5. The Lesch-Nyhan Syndrome is an inherited disorder associated with
a complete deficiency of an enzyme of purine metabolism, which is:
a. HGPRTase.
b. Aspartate carbamayl transferase.
c. Purine nucleotide phosphorylase.
d. Xanthine oxidase.
6. In humans, the end product of catabolism of purines is: a.

-alanine.
b. Allantoin.
170

c. Urea.
d. Uric acid.
III. True / False
1. The end product of purine metabolism in humans is
Allantoin.
2. Gout is a disease that results from decrease of uric acid in blood.
3. Urate crystals deposited in soft tissues as a result of the high solubility of uric acid.
4. Allopurinol is a non-competitive inhibitor of Xanthine
oxidase.
5. Allopurinol is a competitive inhibitor of PRPP synthase.
171

LECTURE # 37 : Pyrimidine Nucleotide Metabolism
Student Notes:
DEPARTMENT: Clinical Biochemistry .
TUTOR:
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1- List the different sources of atoms of pyrimidine ring.
2- List the main steps of de novo synthesis of pyrimidine.
4- State the rate limiting step of de novo synthesis of pyrimidine.
5. Outline synthesis of thymine nucleotides.
6. Outline regulation of pyrimidine nucleotides biosynthesis.
8. List synthetic inhibitors of pyrimidine synthesis and their sites of effect.
9. Describe salvage pathway of pyrimidine synthesis.
10. Discuss catabolism of pyrimidine nucleotides.
11. List end products of pyrimidine catabolism.
172

12. Discuss the biochemical basis of orotic aciduria: causes, classification and treatment.
13. Discuss the formation of dNTP .

De novo synthesis of Pyrimidine
Nucleotides.

Synthesis of Thymine Nucleotides.

Clinical Relevance of tetrahydrofolate.

Regulation of pyrimidine synthesis.

Clinical considerations of Pyrimidine
Catabolism:
-
Orotic aciduria.
-
Chemotherapeutic agents.

Salvage of pyrimidine nucleotides.

Catabolism of pyrimidine nucleotides.

Formation of Nucleoside Di- and
Triphosphates

Formation of Deoxyribonucleotides

Regulation of dNTP formation.

Interconversion of the Nucleotides.
In pyrimidine synthesis, the rate limiting step is catalyzed by cytosolic carbamoyl phosphate synthase II in contrast to the mitochondrial carbamoyl phosphate synthase I that catalyzes the committed step in urea cycle. In contrast to purine synthesis the pyrimidine base is formed first then added to ribose phosphate to give the nucleotide.
In addition, the defect in pyrimidine metabolism is due to deficiency of enzymes that present in the synthetic pathway. As a result the proper treatment involves the oral administration of the required intermediate after the enzymatic block. Accumulation of orotic acid is not harmful as it is water soluble
173

and properly excreted by the kidneys.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about Pyrimidine nucleotides metabolism . You can access the CD-
ROM during your spare time.
16http://www.biochem.med.umich.edu/bioc hem/courses/bc452/downloads/nucleotide s/nucleotide_metab_1.ppt
17www.rpi.edu/~bellos/ Nucleotide s_revised.
ppt
18http://www.ucd.ie/indmicro/patrick2/BL ec1(2004).ppt
19http://www.pharmacy.umn.edu/img/asse ts/12622/lectures1and2.ppt
20http://www.bdid.com/oa.htm
6- Clinical Question: d) Clinical Case:
174

I. Short Questions:
1. Outline the rate-limiting step for pyrimidine synthesis and its regulation.
2. Define the role of uridine monophosphate
(UMP) in nucleotide metabolism.
3. Define orotic aciduria, its symptoms, causes and treatment.
4. What is the significance of ribonucleotide reductase and how is it regulated?
II- MCQs:
1. The first step of pyrimidines biosynthesis is the synthesis of
carbamoyl phosphate catalyzed by:
a. Carbamoyl phosphate synthase I.
b. Carbamoyl phosphate synthase II.
c. PRPP synthase.
d. Phosphoribosyl transferase.
2.
A diet rich in Uridine is the treatment for:
a. Gout.
b. Orotic aciduria.
c. Chronic renal failure.
d. Steatorrhea.
3. Orotic aciduria:
a. Is characterized by accumulation and urinary excretion of
uric acid.
b. May develop in the course of other disease.
c. Resulted from defect of the enzyme orotate phosphoribosyl
transferase. a.
Allopurinol is used in the treatment of this disease.
III. True / False
1. The rate limiting step of pyrimidine synthesis is catalyzed by carbamoyl
phosphate synthase I.
175

2. In pyrimidine nucleotides synthesis, the pyrimidine ring is completed before a
ribose-5-P is added.
3. Carbamoyl-P and aspartate are the precursors of the six atoms of the pyrimidine
ring.
4. Orotic aciduria is a disorder of pyrimidine catabolism.
5. Oral uridine is the treatment of orotic aciduria.
176

LECTURE # 38 : Structure of Nucleic Acids
TUTOR:
TEACHING LOCATION: Auditorium
.
By the end of this lecture you will be able to:
1.
Explain the central dogma of molecular biology.
2.
Define the main functions of nucleic acids.
3.
Identify the structural components of deoxyribonucleic acid (DNA).
4.
Define primary structure of
DNA..
5.
Define secondary structure of
DNA..
6.
List the structural characteristics of Watson and
Crick model of DNA secondary structure.
7.
Differentiate between different forms of DNA..
8.
Define nucleases and differentiate between exo- and endonucleases.
9.
List factors causing DNA denaturation.
177


Central Dogma of Molecular Biology.

Structure and function of nucleic acids.

Structural components of DNA:
Bases, Nucleosides and
Nucleotides.

Primary structure of DNA.

Watson and Crick model of DNA secondary structure.

Different forms of DNA structure.

Nucleases and their specificity.

Denaturation of DNA and melting temperature.
All life on earth uses nucleic acids for storage of genetic information. With the exception of some viruses, the biomolecule utilized for information storage is deoxyribonucleic acid (DNA). This molecule is remarkably well suited for its task because of its chemical stability and its ability to encode vast amounts of information using a simple four-letter code.
The flow of genetic information in all cells is always exclusively one way: DNA

RNA

Protein (central
Dogma of molecular biology).
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Supplementary Texts And Resources:
Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about Structure of DNA.
You can access the CD-ROM during your spare
178

time.
1. http://www.accessexcellence.org/AE
/AEC/CC/DNA_structure.html
2. http://www.johnkyrk.com/DNAanato my.html
3. http://molvis.sdsc.edu/dna/index.ht
m
4. http://www.moleculesinmotion.com/
5. http://www.pwc.k12.nf.ca/wadey/biot ech/dna1.swf
6. http://www.rothamsted.bbsrc.ac.uk/n otebook/courses/guide/dnast.htm
7. http://www.ncc.gmu.edu/dna/index.h
tm
8. http://www.dnaftb.org/dnaftb/ a.
Write an essay on restriction enzymes and their applications in recombinant DNA technology. b.
Write on the applications of DNA denaturation in molecular biology.
I. Short Questions:
1. List the different characteristics of the secondary
structure of DNA.
2. Give an account about nucleases and their applications.
3. What is meant by DNA denaturing.
4. Define each of the following:
a. DNA denaturation.
b. The melting point of DNA.
c. Exonucleases.
5.
List three causes of DNA denaturation.
6.
List forces affecting DNA double
179

helix stability.
II. MCQs:
1. Which statement is true about the double helix:
a. Heating causes the strands to separate
(denature).
b. GC pairs involve three hydrogen bonds.
c. Purine pairs with pyrimidine.
d. All of the above.
2. If a DNA molecule is composed of 40%
(T) what percentage of guanine would be expected:
a. 10%.
b. 20%.
c. 40%.
d. 80%.
3. All of the following are true about DNA
EXCEPT:
a. Guanine usually pairs with cytosine and thymine with adenine.
b. A double helix formed of two antiparallel strands.
c. The sugar-phosphate backbone is positively charged.
d. Base stacking stabilizes the double helix.
4. Nucleases are enzymes that catalyze cleavage of:
a. Peptide bond.
b. Glycosidic bond.
c. Hydrogen bond.
d. Phosphodiester bond.
5. All of the following statements regarding the Watson-Crick
"B" form of DNA are true EXCEPT:
180

a. Two chains are coiled around a common axis forming a right-
handed helix.
b. The bases are found on the outside of the helix and the sugar
phosphate backbone on the inside.
c. The two chains run in opposite directions.
d. Adenine is always paired with thymine, guanine with cytosine.
III. True / False
3.
In DNA double helix, the alternating sugar phosphate units form the backbone while the nitrogenous bases are projecting to the outside.
4.
Exonucleases cleave phosphodiester bonds located in the interior of polynucleotides.
5.
The two strands of DNA double helix are held together by the complementary base pairing through specific hydrogen bonds.
6.
Melting Temperature (Tm) is the temperature at which the two strands of DNA double helix is completely ruptured and separated.
5. Each DNA strand has two terminals one end has a free
phosphate group attached to 5`hydroxyl group of the
terminal pentose and the other end has a free 3`-hydroxyl
group.
181

LECTURE # 39: O rganization of eukaryotic chromatin
TUTOR:
.
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1- Define genome structure.
2- State the differences between viral, prokaryotic and eukaryotic genome structures.
3- Describe the role of histones in DNA packaging.
4- Describe nucleosome structure.
5. Demonstrate the different levels of DNA packaging.

Genome structure in different species.

Genome structure of viruses.

Genome structure of prokaryotes

Genome structure of eukaryotes.

Human genome.

Organization of eukaryotic DNA.

Role of histones in packaging of
DNA.

Nucleosomes and polynucleosomes.

Different levels of organization of eukaryotic DNA.
DNA is usually much larger than the size of the cell containing the DNA. For example, a single diploid human cell contains 6X10 9 bp of
182

chromosomal DNA packaged in 46 chromosomes. The counter length of this DNA approaches 2 meters, all of which is contained within a nucleus, about 10
 m in diameter.
Hence, the DNA of all organisms must be exquisitely packaged.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about Packaging of DNA You can access the CD-ROM during your spare time.
1. http://www.dnaftb.org/dnaftb/
2. http://learn.genetics.utah.edu/units/basics/ tour/
3- http://www.biologie.unihamburg.de/b-online/library/catremoved/u4aos1p2.html
4- http://www.biologie.unihamburg.de/b-online/library/catremoved/u4aos1p2.html
5.
http://www.sciencemuseum.org.uk/ex hibitions/lifecycle/152.asp
6.
http://pulse.pharmacy.arizona.edu/10t h_grade/dawn_new/science/dna_genes.
html
7.
http://morgan.rutgers.edu/MorganWe
183

bFrames/Level1/Page1/p1.html
8.http://www.ndsu.nodak.edu/instruct/ mcclean/plsc431/eukarychrom/eukaryo
3.htm
I. Short Questions:
1. Define each of the following:
a. Histones.
b. Nucleosome structure.
2. List the different levels of DNA packaging.
II. MCQs:
1. All of the following are true about eukaryotic genes EXCEPT:
a. Most of eukaryotic genes are discontineous.
b. They are always overlapping.
c. Coding sequences are unique and non repetitive. d. They contain a regulatory sequence and a coding sequence.
2. Regarding nucleosome structure, which of the following
statements is correct? a. Nucleosomes are spherical structures comprised of RNA,
nonhistone proteins and histone proteins.
b. The major histones comprising nucleosomes are H1and H4. c.
Nucleosomes permit efficient packing of
DNA to about one seventh its normal length. d.
The histone associated with the DNA in the linker region holding nucleosomes is called H2A.
e. All of the above.
184

3. The following statements describes both human and bacterial
DNA EXCEPT:
a. The DNA occurs physiologically as nucleosome complexes.
b. The DNA contains major and minor grooves.
c. The DNA consists of an antiparallel duplex.
d. The DNA contains equal molar fractions of adenine and thymine. e.
The DNA contains equal molar fractions of guanine and cytosine.
4. Histones are small basic proteins that: a. Nucleosome core consists of spherical structures comprised of
DNA surrounded by H3. b.
The major histones comprising nucleosomes are H1and H4. c.
Permit efficient packing of DNA to about one seventh its normal length. d.
The histone associated with the DNA in the linker region holding nucleosomes is called H2A.
e. All of the above.
5. The third level of DNA packaging: a. Nucleosomes, joined by “linker” DNA in presence of H1 form a
polynucleosome.
b. It is by coiling the 30 nm fiber into twisted-looped structure
attached to a protein scaffold in the form of rosettes.
c. It is by formation of nucleosomes in which a segment of the DNA
double helix is wound nearly twice around a histone core.
d. The histone core consists of two molecules each of H2A, H2B,
H3, and H4.
e. All of the above.
185

III. True / False:
1. Human immuno-deficiency virus
(HIV) is RNA virus; its genome is formed of two copies of dsRNA.
2. Prokaryotic genome consists of one single double-stranded supercoiled circular chromosome
3. Plasmid DNA is small and circular extra chromosomal DNA molecules that present in bacteria and undergoes replication that is always synchronized to chromosomal division.
4. Human genome consists of 46 chromosomes that contain about 120,000 genes coding for about 120,000 proteins.
5. Most of the eukaryotic genes are continuous sequences and genes
are usually overlapping.
186

LECTURE # 40 : DNA sequence and function
TUTOR:
.
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1- Compare between prokaryotic genes and eukaryotic genes.
2- Differentiate between the different types of eukaryotic DNA sequences.
3. Identify functions of different types of eukaryotic DNA sequences.

Prokaryotic gene structure.

Eukaryotic gene structure.

Single-copy DNA.

Repeated DNA sequences:

Moderately reiterated DNA.

Highly reiterated DNA.

Inverted repeat DNA.
The size and average base composition of DNA vary widely between species. The property that makes the DNA of a species unique is the nucleotide sequence. The human genome project offers a complete sequence of human genome. In contrast to prokaryotic genome, eukaryotic genome contains repetitive sequences that are repeated anywhere from a few times, for certain coding genes, to million of times per
187

genome for certain simple, relatively short, sequences.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss
3. Human Molecular Genetics, Strachan
T. & Read, A.P. BIOS Scientific
Publishers
Limited.



You have the opportunity to watch the CD-
ROM about DNA Sequence and Function.
You can access the CD-ROM during your spare time.
1- http://www.webbooks.com/MoBio/Free/Ch3G.htm
2- http://www.neuro.wustl.edu/NEUROMUSC
ULAR/mother/dnarep.htm#gengenom
3- http://library.lanl.gov/cgibin/getfile?00418723.pdf
4- http://www.math.umd.edu/~ssindi/mainthe sis.pdf
5- http://www.pubmedcentral.nih.gov/articler ender.fcgi?artid=324220
6. http://en.wikipedia.org/wiki/Alu_sequence
7. http://en.wikipedia.org/wiki/Category:Repe
188

titive_DNA_sequences
I. Short Questions:
1. List the different classes of eukaryotic repeated DNA sequences.
2. List different types of single copy DNA sequences.
II. MCQs:
1. The unique sequence DNA includes all the following EXCEPT: a. Alu repeats. e.
Most coding genes. f.
Pseudogenes. g.
Regions flanking genes. f.
Introns.
2. The Alu repeat : a. Involves most genes.
b. Is 300 base pairs long, present in about
500,000 copies per haploid genome c. Is found only within genes in introns. d. Is the main component of Pseudogenes. h.
Present mainly in centromeres of chromosomes.
e. Present mainly in telomeres.
3. Microstellit sequences :
a. Sequences of 2-5 base pairs repeated up to 50 times.
b. Exists as both dispersed and grouped tandem arrays.
c. Most commonly found as dinucleotide repeats of AC.
d .The GT repeats range in size from about
20 to 60 base pairs.
e. All of the above.
189

III. True / False
1. Most of the prokaryotic genes are continuous sequences and genes are usually
overlapping.
2. Eukaryotic genes consist of coding sequences (exons) interrupted by intervening
sequences (introns).
3. Pseudogenes are sequences of DNA that have significant nucleotide homology to
a functional gene but that contain mutations that prevent gene expression.
4. As distinct from eukaryotes, the DNA of prokaryotes contains multiple copies of
repetitive sequences.
5. In human DNA, at least 25-35% of the genome consists of repetitive sequences.
190

LECTURE # 41 : Replication of DNA (DNA Synthesis)
TUTOR:
.
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
6- State the role replication in transfer of genetic information.
7- List the common features of replication.
8- List the different components of replication.
9- Define replication fork and its role in replication.
10- Explain the different steps of replication.
11- Distinguish between prokaryotic and eukaryotic replication.
12- List the different activities of DNA polymerases.
13- State the different DNA polymerase and their role in replication in both prokaryotes and eukaryotes.
14- Explain the mechanism of DNA proofreading of newly synthesized DNA strands.
15- Discuss the role of topoisomerases during replication.
16- Explain the role of telomerases in replication.
17- List the different inhibitors of replication and their site of effect.

Principles of replication.

Components of DNA replication.
191


Origin of replication.

Prokaryotic replication:
H. Single origin of replication.
I. Proteins required for DNA separation.
J. Prokaryotic DNA polymerases.
K. Synthesis of Leading and
Lagging strands.

Eukaryotic replication:
L. Multiple origin of replication.
M. Eukaryotic DNA polymerases.
N. Synthesis of Leading and lagging strands.

Proofreading of newly synthesized
DNA strands.

DNA Topoisomerases.

Telomerases.

Inhibitors of replication.
Remember that DNA, the storehouse of genetic information, is required for the storage and expression of genetic information. This genetic information is copied and transmitted to daughter cells through DNA replication.
The DNA contained in a fertilized egg encodes the information that directs the development of an organism. This development may involve the production of billions of cells. Each cell is specialized, expressing only those genes that are required its function. Therefore, DNA must be able to not only replicate precisely each time a cell divides, but also to have the information that it contains be selectively expressed.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th
192

edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about Replication of DNA . You can access the CD-ROM during your spare time.
21http://www.biostudio.com/d_%20Replic ation%20of%20a%20Chromosome.htm
22http://www.biostudio.com/d_%20DNA%
20Replication%20Nucleotide%20Polym erization.htm
23http://www.biostudio.com/d_%20DNA%
20Replication%20Coordination%20Lea ding%20Lagging%20Strand%20Synthe sis.htm
4- Inhibitors of DNA replication are used clinically for treatment of different diseases. In the light of the above, deduce the difference between inhibitors of prokaryotic replication and eukaryotic replication in clinical applications.
Clinical Question
I. Short Questions:
1. List different characteristics of DNA replication.
193

2. List different types of eukaryotic DNA polymerases and their functions.
3. Define:
a. Origin of replication.
b. Semiconservative replication.
4. Give function of the following:
a. Helicase.
b. Single strand DNA-binding proteins.
c. DNA-derpendent DNA polymerase.
5. List different characteristics of origin of replication.
6. Compare between prokaryotic DNA polymerases.
7. List the steps of eukaryotic DNA replication.
8. List three inhibitors of DNA replication.
II- MCQs:
1. All of the following are true about eukaryotic DNA replication EXCEPT:
a. It is semiconservative.
b. It occurs in the 3

5

direction.
c. Both strands act as a template simultaneously. d. It is bidirectional.
2. Which statement is TRUE about DNA replication: a. It occurs on one strand only
b. It is catalyzed by RNA polymerase.
c. It occurs in the 3

5

direction.
d. It is semiconservative
3. DNA replication requires all the following EXCEPT:
a. RNA polymerase
b. A short RNA primer.
c. Deoxynucleoside triphosphates. d. DNA template.
4. In replication, separation of the double
194

strands of DNA is catalyzed by:
a. Helicase.
b. Primase.
c. DNA polymerase. d. RNA [polymerase.
5. Single stranded DNA (ssDNA) binding proteins play a role in the following
EXCEPT:
a. Packaging of DNA.
b. Bind to ssDNA
c. Keep the two DNA strands separated.
d. Protects ssDNA from digestion by nucleases.
6. During DNA replication, the sequence 5′-
TpApGpAp-3′ would produce which
of the following complementary sequence:
a. 5′-TpCpTpAp-3′.
b. 5′-ApTpCpTp-3′.
c. 5′-UpCpUpAp-3′.
d. 5′-GpCpGpAp-3′.
III. True / False
7Replication process involves replication of certain genes of DNA.
8- Ligase enzyme plays a major role in the separation of the two DNA strands for replication.
9- In prokaryotes, replication starts at multiple sites that are rich in AT sequences.
10- Single Stranded DNA binding proteins bind to the single stranded
DNA keeping the 2 strands separated.
11- DNA polymerases utilizes ribnucleoside triphosphates for the synthesis of the new strands.
195

LECTURE # 42 : DNA Repair
Student Notes:
DEPARTMENT: Clinical Biochemistry .
TUTOR:
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1- Define gene mutations.
2- List the different causes of mutations.
3- Specify the different types of mutations.
4- Describe the mechanism of DNA repair.
5-Indicate the consequences of defective DNA repair mechanism.

Gene mutations.

Causes of mutations.

Types of mutations.

DNA repair mechanism.

Examples of defective DNA repair mechanism.
Remember that despite the elaborate proofreading system employed during DNA synthesis, mutations can occur. In addition,
DNA is constantly being subjected to environmental insults that cause the alteration or removal of bases. Bases are also altered or lost spontaneously from mammalian DNA.
If the damage is not repaired, a permanent mutation may be introduced that can result in any of a number of deleterious effects.
Luckily, cells are remarkably efficient at repairing the damage done to their DNA.
196

1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about DNA Repair . You can access the CD-ROM during your spare time.
1- Clinical Question: a) Clinical Case:
I. Short Questions:
1. List different types of mutations.
2. DNA repair is an important process:
a. List the different causes of mutations.
b. Outline steps of DNA repair.
II. MCQs:
1. What will be the effect of a single base pair deletion in the middle of a DNA
sequence coding for a certain protein?
197

a. A protein with a single amino acid substitution in its centre.
b. The gene will not be transcribed and no protein will be produced.
c. The amino half of the protein will have a normal sequence.
d. The carboxyl half of the protein will have a normal sequence.
e. DNA polymerase will repair the damage.
2. Which one of the following enzymes catalyzes the elimination of nicks or
discontinuities in DNA that occur during
DNA repair or in the linking together of
Okazaki fragments? a. AP exonuclease.
b. DNA gyrase.
c. DNA ligase.
d. Topoisomerase I.
e. Topoisomerase II.
3. Xeroderma pigmentosum results from a deficiency in: a. Collagen synthesis.
b. DNA replication.
c. DNA repair.
d. Bilirubin conjugation.
e. A tumor suppressor.
III. True / False
1. Mutations are permanent changes in a
DNA sequence.
2. Frame shift mutation results from a single base substation.
3. Xeroderma Pigmentosum is a rare autosomal recessive disease due to
defective repair.
4. The DNA repair mechanism involves recognition of the lesion by an
exonuclease, which cleaves the damaged strand to form a nick.
5. Excision of damaged DNA is catalysed
198

by an excision “3′  5′ ”
exonuclease.
199

LECTURE # 43 : Structure of Ribonucleic Acid (RNA)
TUTOR:
.
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1-
2-
3-
4-
5-
6-
7-
Identify the structural components of ribonucleic acid (RNA).
List the different types of RNA.
Explain the structural characteristics of rRNA.
Explain the structural characteristics of tRNA.
Explain the structural characteristics of rRNA.
Differentiate between the functions of different types of RNA.
Illustrate the importance of interaction between the three different RNAs in protein synthesis.

Structure of Ribonucleic Acid (RNA).

Types of RNA:
O. Ribosomal RNA.
P. Transfer RNA.
Q. Messenger RNA.
R. Nuclear RNA.
S. Mitochondrial RNA.
RNA molecules play different roles in cellular information transfer. All three types of
RNA; ribosomal RNA, transfer RNA and messenger RNA interact together for the process of protein synthesis. Ribosomes carry out protein synthesis. Transfer RNAs
200

transfer specific amino acids to ribosomes.
Messenger RNAs carry information from the
DNA to the cellular protein synthetic machinery. Other RNAs in the cell are important in RNA processing and in protein export from the cell.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about RNA Structure . You can access the CD-ROM during your spare time.
1- Clinical Question a)
2- Clinical Case
I. Short Questions:
1. Enumerate the major differences
201

between DNA and RNA.
2. List the different characteristics of tRNA and function of each.
3. List the structural features of mRNA.
II. MCQs:
1. RNA is found mainly in:
a. Cytoplasm.
b. Nucleus.
c. Mitochondria.
d. Lysosomes.
2. The sugar present in RNA is:
a. Ribose.
b. Ribulose.
c. Deoxyribose.
d. Glucose.
3. RNA has the following features
EXCEPT:
a. Contains purine bases.
b. Contains pyrimidines bases.
c. Contains phosphate.
d. Is double stranded helix.
4. All of the following are present in RNA
EXCEPT:
a. Deoxy ribose.
b. Ribose.
c. Adenine.
d. Cytosine.
5. The DNA & RNA share the following features EXCEPT:
a. Both contain purine bases.
b. Both contain pyrimidines bases.
c. Both contain phosphate.
d. Both are double stranded helix.
202

6. If a sample of mRNA is 30 mole % G, the mole % A: a. is greater than 30.
b. is less than 30.
d. cannot be ascertained from this information.
e. is also 30.
7. Acceptor arm of tRNA is characterized by the following:
a. Contains three specific bases
(anticodon) that is complementary to mRNA. b. contains from 3 to 12 bases and it is the major site for variation in tRNA. c. It terminates at its 3` OH end by a specific sequence formed of CCA. d. Contains the unusual base dihydrouracil so termed the D-loop.
e. Contains the unusual thymine and pseudouridine bases.
III. True / False
1. The main function of mRNA is the transport of amino acids to the ribosome
for synthesis of proteins.
2. The anticodon loop of tRNA plays a key role in translation by pairing with the
complementary codon of mRNA.
3. Each mRNA molecule carries a message to represent the genetic information
of a specific gene for the synthesis of a specific protein.
4. RNA molecules are synthesized in a process termed replication where the
sequence of bases in each RNA molecule is controlled by base sequence in
both strands of DNA.
5. Eukaryotic 80S ribosomes are formed mainly of two main nucleoprotein
subunits of unequal size, the 60S subunit and the 40S subunit.
203

204

LECTURE # 44 -a : Transcription of DNA (RNA Synthesis)
TUTOR: .
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1. State the role transcription in expression of genetic information.
2. State the general principle of transcription.
3. List the different components of transcription.
4. Explain structure and functions of RNA polymerase.
5. State the functions of different types of
RNA polymerases.
6. Explain the different phases of transcription.
7. Discuss the steps of eukaryotic mRNA synthesis.
8. State the role of promoter sequence in transcription.
9. Explain the role of transcription factors in transcription.
10.
List the different inhibitors of transcription and their site of effect.

General principles of transcription.

Eukaryotic transcription.

Components of transcription.

RNA polymerases.

Types of RNA polymerases.

Transcription phases:
T. Initiation.
205

U. Elongation.
V. Temination.

Synthesis of mRNA.

Transcriptional gene regulatory elements.

Inhibitors of transcription.
Remember that gene expression involves both transcription and translation processes.
Transcription is the process by which the information stored in DNA for certain protein sequence is copied from one strand of a template sequence into the sequence of mRNA.. This mRNA then acts as a template for the synthesis of proteins. The sequence of nucleotides on DNA specifies the sequence of nucleotides on mRNA, which in turn specifies the sequence of amino acids in proteins.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about Transcription . You can access the CD-ROM during your spare time.
206

1. www.uhmc.sunysb.edu/som/courses/mgac/Lects.%20
16
&.
2. www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/26transl.
ppt
3. http://www.biostudio.com/d_%20Transcription.ht
m
4. http://207.207.4.198/pub/flash/26/transmenu_s.s
5. wf
Clinical Question:
1- Clinical Case
I. Short Questions:
1. Tabulate the similarities and differences between eukaryotic replication and
Transcription (function, site, enzyme, direction, requirements).
2. In a tabular form compare between
DNA-polymerase and RNA-polymerase
(function, activity, requirements).
3. List the function of

subunit of RNA polymerase.
4. List different types of RNA polymerases and their functions.
II. MCQs:
1. Transcription of a gene in human cells:
a. Always begins at an AUG (codon which codes for methionine).
b. Does not require local unwinding of two
DNA strands.
c. Always “reads” the DNA template
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strand in the 3′ to 5′ direction.
d. Occurs on both strands simultaneously.
2. Synthesis of RNA from a DNA template requires: a. ddA, ddC, ddG and ddT nucleotides.
b. A, C, G and U triphosphate nucleotides.
c. Reverse transcriptase.
d. dA, dC, dG and dT triphosphate nucleotides
e. Restriction endonucleases.
3. All of the following is true about RNA polymerase EXCEPT:
a. It requires a primer.
b. It has a 3

5

polymerase activity.
c. It utilizes ribonucleoside triphosphates.
d. Is a polymeric enzyme.
4. TATA box is recognized by the following subunit of RNA polymerase: a. α subunit. b.
β
subunit.
c. Core enzyme.
d.

subunit.
5. TATA box:
a. Determines the start of trascription.
b. Determines the frequency of transcription.
c. Occurs down stream of the structural gene.
d. consists of 20 bases.
6. The

subunit of RNA polymerase:
a. Enables RNA polymerase to recognize and bind to the promoter.
b. Determines which strand is copied.
c. Determines the start of transcription.
d. All of the above.
7. During RNA synthesis, the DNA template sequence 5′-ApTpGpCp-3′
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would be transcribed to produce which of the following RNA sequences:
a. 5′-TpApCpGp-3′.
b. 5′-GpCpApTp-5′.
c. 5′-ApUpCpGp-3′.
d. 5′-GpCpApUp-3′.
8. Concerning TATA box (Hogness box):
a. It encodes repressor protein.
b. It binds to anticodon.
c. It regulates translation.
d. It binds to RNA polymerase.
9.
All of the following are true for both
DNA polymerase and RNA polymerase
EXCEPT: a. Both require a template.
b. Both reactions produce pyrophosphate as a product.
c. Both add 5' nucleotides to 3' hydroxyl groups.
d. both utilize nucleotide triphosphates as substrates.
e. Both require a primer.
III. True / False
1. The first step in mRNA synthesis is the binding of the TF IID to the TATA
Box.
2. The CAAT box and the GC box determine the frequency of transcription.
3. The complex of RNA polymerase,
DNA template and new RNA transcript
is called transcription bubble.
4. In transcription, RNA polymerase utilizes deoxyribonucleoside
triphosphate and releases pyrophosphate.
5. Initiation of transcription involves the binding of RNA polymerase to the
promoter regi
LECTURE # 44-b : Posttranscriptional processing and Regulation of
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Gene Expression
Student Notes:
DEPARTMENT: Clinical Biochemistry .
TUTOR:
TEACHING LOCATION: Auditorium
By the end of this lecture you will be able to:
1. List postranscriptional processing of RNA.
2. Explain postranscriptional processing of mRNA.
3. List the different levels of regulation of eukaryotic gene expression.
4. Differentiate between the different levels of regulation of gene expression.
5. Identify the basal expression elements and their role in regulation of gene
expression.
6. Identify the regulated expression elements and their role in regulation of gene
expression.
7. Explain the role of alternative splicing in regulation of gene expression.
8. Explain the role of polyadenylation in regulation of gene expression.

RNA processing.
 mRNA processing.

Levels of eukaryotic Gene Expression
Regulation: a. Alteration of Gene Content: i. Gene Amplification. ii. Gene Diminution. iii. Gene Rearrangement or
Recombination. b. Transcriptional regulation: i. Chromatin Remodeling.
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ii. DNA Regulatory Region:
- Basal Expression
Elements: TATA box, and CAAT box or
GC
box.
- Regulated Expression
Elements: Enhancers, Silencers and
other regulatory elements. iii. DNA Regulatory Factors. c. Posttranscriptional Regulation: i. Alternative Splicing. ii. Regulation of RNA Stability.
The process of transcription involves transcription of the whole gene to give primary transcript. However, most eukaryotic genes are interrupted by introns that are not coding for proteins. Posttranscriptional modification of RNA involves many chemical modifications along with excision of primer sequences and ligation of the exons (coding sequences) together to give mature mRNA ready to be transferred to the cytoplasm where protein synthesis occurs on ribosomes.
While the whole set of chromosomes are present in every cell, genes expression in different cell types vary widely according to the activity of individual cells. Hence, gene expression is finely controlled through different mechanisms of gene regulation.
1. Required Texts And Resources: Lippincott
Illustrated Reviews, 3 rd edition, Champe &
Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition,
Wiley-Liss


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
You have the opportunity to watch the CD-
ROM about RNA processing . You can access the CD-ROM during your spare time.
1http://highered.mcgrawhill.com/sites/007243
2http://207.207.4.198/pub/flash/26/transmenu
_s.swf
3-
7316/student_view0/chapter15/animations.h
tml#
1. Clinical Question:
2. Clinical Case
I. Short Questions:
1. List the different posttranscriptional modifications of RNA.
2. List the different posttranscriptional modifications of mRNA.
3. Define the following and give function: a.
The TATA box. b.
Capping of mRNA.
c.
Polyadenylation of mRNA.
d.
Regulated expression elements.
4. List the different levels of regulation of eukaryotic gene expression.
5. Define:
a. Basal regulatory elements.
b. Enhancer.
c. Silencer.
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II. MCQs:
1. Regulation of eukaryotic gene expression involves:
a. Alteration of gene content. b. Transcriptional regulation. c. Posttranscriptional regulation. d. All of the above. e. None of the above.
2. Gene expression: a. Shows no relation with the tissue activities. b. Is the same in all tissues due to the same chromosome structure. c. Involves replication of the whole genome. d. Differ in different tissues due to the different chromosome structure. e. Differ in different tissues according to the different tissue activities.
3. Basal expression elements involves:
a. TATA box. b. Enhancers. c. Silencers. d. Other regulatory elements. e. Poly A tail.
4. Histone acetylation at their N-terminal
(lysine):
a. Increases the histone positive charges and decreases the binding affinity of
histones with the negatively charged DNA.
b. Increases the histone positive charges and increases the binding affinity of
histones with the negatively charged DNA. c. Reduces the histone positive charges and decreases the binding affinity of histones
with the negatively charged DNA. d.
Reduces the histone positive charges and increases the binding affinity of histones
with the negatively charged DNA. e.
None of the above.
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III. True / False
1. Methylation of cytosine of DNA converts the active euchromatin into inactive
heterochromatin.
2. Enhancers interact with gene regulatory proteins or trans-factors and decrease
the rate of expression.
3. Basal expression elements contains TATA box and CAAT box or GC box.
4. TATA box specify the frequency of initiation.
5. The length of poly A tail determines the half life time of mRNA.
214

LECTURE # 45 : Translation of RNA (Protein Synthesis)
TUTOR:
TEACHING LOCATION: Auditorium
.
By the end of this lecture you will be able to:
1. State the principle of translation (protein synthesis).
2. Define genetic code.
3. Explain the relation between genetic code and amino acid sequence in the process of protein
synthesis.
4. Define initiation codon and stop codons.
5. List the steps involved in protein synthesis.
6. Describe activation of amino acids (synthesis of amino acyl-tRNA).
7. Discus the steps of polypeptide chain synthesis.
8. Define the role of different initiation factors in the initiation of protein synthesis.
9. Describe the interaction between different types of
RNA in the process of protein synthesis.
10. Define the role of different elongation factors in the elongation of polypeptide chain.
11. State the role of peptidyl transferase in the process of protein synthesis.
12. Discus the process of termination of protein synthesis and its regulation.
13. Explore the role of polyribosome in enhancing the synthesis of protein.
14. List the different posttranslational modifications of proteins.
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
Principle of Protein Synthesis.

Genetic Code.

Protein Synthesis:
I. Synthesis of Aminoacyl-tRNA
(Activation of Amino Acids).
II. Synthesis of Polypeptide Chain
(translation):
1. Initiation:
1) Ribosomal
Dissociation.
2) Formation of the 43S
Preinitiation Complex.
3) Formation of 48S
Initiation Complex.
4) Formation of the 80S
Initiation Complex.
- Regulation and
Control of Initiation.
2. Elongation:
1) Binding of aminoacyltRNA to the A Site.
2) Peptide Bond
Formation.
3) Translocation.
3. Termination

Polyribosomes.

Posttranslational Processing of
Proteins: iii. Trimming. iv. Covalent Alteration.
Translation is the second step by which gene expression leads to protein synthesis.
Protein synthesis involves interaction of the three main types of RNA. The sequence of codons in mRNA specifies the order of amino acids in a protein. Protein synthesis takes place on the ribosomes by the aid of tRNA. This process involves activation of amino acids then synthesis of polypeptide chain. In addition,
216

many proteins requires posttranslational modifications to give rise to the functional protein.
1. Required Texts And Resources:
Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Textbook of Biochemistry with Clinical
Correlations, Thomas M. Devlin, 5th edition, Wiley-Liss



You have the opportunity to watch the CD-
ROM about Translation.
You can access the CD-ROM during your spare time.
1.
http://highered.mcgrawhill.com/sites/007243
7316/student_view0/chapter15/animations.ht
ml# www.uhmc.sunysb.edu/som/courses/mgac/Lect
2.
1 & s.%2016
3.
http://www.biostudio.com/demo_freeman_p rotein_synthesis.htm
4.
http://207.207.4.198/pub/flash/26/transmenu_s
.swf
Clinical Question:
217

Clinical Case
I. Short Questions:
1.
Enumerate the steps of protein synthesis.
2.
List different initiation factors of protein synthesis and their function.
3.
List the posttranslational modifications of proteins.
II. MCQs:
1. A codon consists of :
a. One molecule of aminoacyl-tRNA.
b. Three successive bases.
c. Four individual nucleotides.
d. Two complementary base pairs.
2. All of the following are requirements for protein synthesis EXCEPT:
a. Ribosomes.
b. mRNA.
c. Aminoacyl-tRNA.
d. Carbamoyl phosphate synthetase I.
3. All of the following are required for protein synthesis EXCEPT:
a. Ribosomes.
b. mRNA.
c. Aminoacyl-tRNA.
d. Primer protein.
4. Activation of amino acids means:
a. Synthesis of peptide bond. b. Translocation of peptidyl-tRNA. c. Synthesis of aminoacyl-tRNA. d. Ribosomal dissociation. e. Hydrolysis of GTP.
5. All of the following is required for protein
218

synthesis EXCEPT: a. mRNA. b. Aminoacyl tRNAs. c. Ribosomes. d. DNA template. e. Peptidyl transferase.
6. Which one of the following choices best completes the following
sentence?
Transfer RNA……..
a. Contains the information necessary for the synthesis of a specific protein.
b. Must exist in at least 20 different forms, one for each amino acid.
c. Is the largest of RNA species.
d. Has little or no secondary structure.
7. Initiation of eukaryotic peptide synthesis requires:
a. The initiation factors.
b. The enzyme RNA polymerase.
c. The enzyme peptidyl transferase.
d. The enzyme translocase.
8. Which one of the following statements about messenger RNA is
INCORRECT?
a. The sugar moiety of mRNA is D-ribose.
b. The polynucleotde chain at mRNA is longer than that of DNA.
c. mRNA exists as single-stranded molecules.
d. mRNA has a 7′-methylguanosine “cap” at its 5′-end.
9. During the process of peptide chain elongation, the growing peptide chain:
a. Is linked to the mRNA.
b. Is linked to the ribosomal RNA.
c. Is not linked at all to any RNA.
d. Is linked to template DNA.
219

e. Is linked to the 3'-OH of the adenosine terminal nucleoside of tRNA.
III. True / False
1 . Different proteins are synthesized in different tissues due to the different
chromosome structure.
2. Phosphorylation of IF-2 prevents formation of 43S preinitiation complex and
blocks protein synthesis.
3. A site is the ribosomal site where tRNA carrying the growing polypeptide chain
binds.
4. Elongation of protein synthesis is a cyclic process and involves synthesis of
Aminoacyl-tRNA.
5. A codon is formed of three specific nucleotides ( or three nitrogenous bases).
220

Lectures #(46) : Recombinant DNA Technology
DEPARTMENT: Clinical Biochemistry
Tutor: Dr.
TEACHING LOCATION: Auditorium
Student Notes:
.
By the end of this topic the student shall be able to:
1. Describe the basic techniques utilized in recombinant DNA.
2. Know the utility of these techniques in diagnosis and research.

Introduction.

Hybridization.

Methods used in labeling nucleotide proteins.

DNA storage.

DNA amplification and cloning.

Cell-based cloning.

Polymerase chain reaction-based
DNA cloning.

Specific methods used in the analysis of DNA.

PCR-based methods.

Other methods for detection of variation in DNA.

DNA sequenc ing
The rapid development of techniques in the field of molecular biology is revolutionizing the practice of medicine.
221

The potential uses of these techniques for the diagnosis and treatment of diseases are vast. Techniques of molecular biology are used in the prevention and treatment of diseases. For example, recombinant
DNA techniques provide human insulin for the treatment of Diabetes. In these 2 lectures some of recombinant DNA techniques will be described as Methods of isolation, amplification and manipulating
DNA involve the use of restriction enzymes, cloning vectors, and PCR.
1. Lippincott Illustrated Reviews, 3 rd edition, Champe & Harvey
2. Reading Handouts will be distributed
3- Lectures and power point presentation will be published on department website: www.kaau.edu.sa/faculties/medicine/dcbcweb
You have the opportunity to watch the CD-
ROM about Extracellular matrix You can access the CD-ROM during your spare time.
As a process of Self Directed
Learning (SDL) search in the internet for web sites related to this subject and write them down:
1-
222

2-
3-
A 70-year-old man presented with back pain and
Joss of weight. Although a non-smoker, he had had several recent chest infections and was increasingly short of breath on exercise. On examination, he was anaemic but there were no other obvious abnormalities.
Investigations
Sodium 130 mmol/L
urea 15.3 mmol/L creatinine 212umol/L calcium 2.75 mmol/L total protein 85g/L albumin 30g/l urate 0.51 mmol/L
ESR >100mm/h haemoglobin 8.5 g/dL
A blood film showed normochromic, normocytic anaemia; rouleaux were present on the blood film and there was increased background staining.
Serum protein electrophoresis revealed a paraprotein in the ^globulin region [Fig. 13.4, pattern b); this was typed by immunofixation and shown to be IgG-K. There was a decrease in the normal yglobulin band. Bence Jones protein was present in the urine and identified as k in type.
Radiological examination showed the typical punched-out lytic lesions of myeloma in the lumbar vertebrae, ribs and pelvis.
What is your comments on this case?
Make internet search about gene
223

therapy. With the aid of figure write 3 to 4 pages on the principles of gene therapy and its applications in human diseases.
I- Short Questions:
1- What are the methods of DNA amplification.
2- Enumerate Medical applications of
PCR
3 - What is meant by DNA finger printing?
II- MCQ:
1- All of the following are desirable features of a good vector EXCEPT:
A. An origin of replication
B.
C.
A gene for selectable marker
A restriction enzyme site for insertion of targets DNA
D. The ability to prevent lysis of the host cell.
III- True / False a- Restriction enzymes are best described by conferring a selective advantage on invading bacteriophage b- Restriction fragment polymorphism analysis isolate a gene that causes an inherited disease.
224

Practical (1) : Enzymes: Amylase activity in serum
Department: Biochemistry
Teaching location: Classroom
By the end of this practical the student will be able to: o Determine the optimum temperature for enzyme activity o Estimate serum amylase activity in the provided sample o Explain the function of serum amylase o Discuss the diagnostic importance of serum amylase

Determination of serum amylase

Explaining the effect of temperature on enzyme activity

Discuss the role of measurement of serum amylase in disease condition

Evaluating the role of amylase in GIT digestion of nutrients
Transferable skills:

Interrelate laboratory data with clinical findings
READING: Laboratory Handouts will be distributed
225

Practical (2) : Enzymes: Estimation of serum alkaline phosphatase
Department: Biochemistry
Teaching location: Classroom
By the end of this practical the student will be able to: o Determine serum alkaline phosphatase o Evaluate the effect of enzyme inhibitors on enzymatic activity o Define Km and Vmax o Show how the Lineweaver-Burk plot can be used to evaluate Km and
Vmax

Determination of serum alkaline phosphatase

The definition of Km and Vmax and the effect of enzyme inhibitors on both of them

Plotting of Lineweaver-Burk plot and its utilization for evaluation of Km and
Vmax

Clinical importance of measurement alkaline phosphatase
Transferable skills:

Interrelate laboratory data with clinical findings

Serum alkaline phosphatase as marker for bone and liver diseases
READING: Laboratory Handouts will be distributed
226

Practical (3) : Enzymes: Serum CK isoenzymes
Department: Biochemistry
Teaching location: Classroo
ة
By the end of this practical the student will be able to: o Define what is meant by isoenzymes o Separate the different CK isoenzymes using electrophoresis o Evaluate the role of CK in tissues o Discuss the role and importance of measuring CK isoenzymes in different diseases conditions

Separation of serum CK isoenzymes

Function of Ck in different tissues

The different isoenzymes of CK and relation to different diseases
Transferable skills:

Interrelate laboratory data with clinical findings

How to use CK isoenzymes in evaluating cardiac and muscle diseases
READING: Laboratory Handouts will be distributed
227

Practical (4) : Serum lactate
Department: Biochemistry
Teaching location: Classroom
By the end of this practical the student will be able to: o Determine blood lactate o Evaluate the importance of determination of serum lactate o To discuss how one enzyme affects different metabolic pathways

Determination of serum lactate

The importance of measurement blood lactate in evaluating children with hypoglycemia

The relation of lactic acidosis to enzyme deficiency such as glucose 6-phosphatase deficiency
Transferable skills:

Interrelate laboratory data with clinical findings
READING: Laboratory Handouts will be distributed
228

Practical (5) : Serum lactate dehydrogenase isoenzymes
Department: Biochemistry
Teaching location: Classroom
By the end of this practical the student will be able to: o Use electrophoresis for separation of LDH isoenzymes o Differentiate between the different LDH isoenzymes (sources and function) o Evaluate the role of LDH isoenzyme in disease diagnosis

Determination of serum LDH isoenzymes using cellulose electrophoresis

The role of LDH in different metabolic pathways

The sources of LDH isoenzymes and its role in diagnosis
Transferable skills:

Interrelate laboratory data with clinical findings

How to use LDH isoenzymes in evaluating cardiac cases
READING: Laboratory Handouts will be distributed
229

Practical (6) : Liver glucose-6-phosphatase deficiency
Department: Biochemistry
Teaching location: Classroom
By the end of this practical the student will be able to: o Determine glucose-6-phosphatase in tissue extract o Discussing the different changes in blood glucose, lactate, pyruvate, fatty acids, ketone bodies and uric acid found in this case o Explaining the basis of other glycogen storage diseases

Determination of glucose-6-phosphatase activity in liver tissues
Transferable skills:

Interrelate laboratory data with clinical findings

Correlation of lactic acidosis with glucose-6-phosphatase deficiency
READING: Laboratory Handouts will be distributed
230

Practical (7) : Plasma glucose estimation & Oral glucose tolerance curve
Department: Biochemistry
Teaching location: Classroom
By the end of this practical the student will be able to: o Estimate plasma glucose o Differentiate between the different methods for measuring plasma glucose o Utilize the result of plasma glucose to diagnose a case of hyperglycemia o Discuss how to monitor a diabetic patient o Draw glucose tolerance curve o Utilize this curve in diagnosis of hyperglycemic case
Blood Ammonia Determination
a- Principle
b- Specimen
c- Reagents
d- Procedures
e- Calculation
4. Case discussion

Determination of plasma glucose

Difference between blood glucose and plasma glucose

WHO classification of hyperglycemia

Drawing the curve using different glucose concentration

Estimation of the different samples (fasting, 60 minutes, 90 minutes and 120 minutes)

The difference between oral glucose and intravenous glucose tolerance curve

Monitoring of diabetic patient
Transferable skills:
231


Interrelate laboratory data with clinical findings

How to utilize plasma glucose for diagnosis and monitoring diabetic patients
READING: Laboratory Handouts will be distributed
232

Practical (8) : Lipid Estimation of plasma total cholesterol & Triacylglycerol
Department: Biochemistry
Teaching location: Classroom
By the end of this practical the student will be able to: o Estimate plasma cholesterol using cholesterol oxidase method o Correlate plasma cholesterol level with cardiac diseases o Utilize biochemical knowledge in treating this patient o Estimate plasma triacylglycerol o Evaluate the role of lipoprotein lipase in lipid metabolism o Differentiate between the different types of hyperlipidemia

Measurement of plasma total cholesterol

Different types of hyperlipidemia

Classification of hypercholesterolemia according to Adult Treatment Panel III
(ATP III) provided by National Cholesterol Education Program (NCEP)

Different types of hyperlipidemia

Classification of hyperlipidemia according to Adult Treatment Panel III (ATP III) provided by National Cholesterol Education Program (NCEP)
Transferable skills:

Interrelate laboratory data with clinical findings

Approach a case of hypercholesterolemia

Approach a case of hyperlipidemia
READING: Laboratory Handouts will be distributed
233

Practical 9: Serum Urea & Ammonia Determination
Department: Biochemistry
Teaching location: Classroom & Biochemistry Laboratory
By the end of this topic the student shall be able to:
1. Knowing the causes of changes in Blood Urea.
2. Determine urea by diacetyl monoxime method
3.determine ammonia by Brthelot reaction
4.Know what is meant by hyperammonemia
1- Introduction.
2. Case History
3. Blood Urea & Ammonia Determination
a- Principle
b- Reagents
c- Procedures
d- Calculation
Transferable skills:
1.Acquire manual skills in handling laboratory equipments
2.Correlate the laboratory findings with theoretical knowledge
READING: Handouts will be distributed before the class
234

Practical 10 : Serum and urinary creatine and creatinine
Department: Biochemistry & Biochemistry Laboratory
Teaching location: Classroom
By the end of this topic the student shall be able to:
1. Determine creatine and creatinine in serum and urine
2. Understand the role of creatine phosphate in muscle
3. Correlate creatine and creatinine level to disease states
1- Introduction.
2. Case History
3. Serum and urinary creatine and creatinine
a- Priniciple
b- Reagents
c- Procedures
d- Calculation
4. Case discussion
Transferable skills:
1.Acquire manual skills in handling laboratory equipments
2.Correlate the laboratory findings with theoretical knowledge
READING: Handouts will be distributed before the class
235

Practical 11 : Serum Alanine aminotransferase (ALT) & Serum Aspartate aminotransferase (AST)
Department: Biochemistry
Teaching location: Classroom & Biochemistry Laboratory
By the end of this topic the student shall be able to:
1. Understand the mechanisms involved in the development of liver damage related to chronic liver disease
2. Understand the principle of the common methods for the measurement of Alanine aminotransferase
3. Learn how to interpret biochemivcal data in relation to a disease state
(in this case chronic liver disease)
1- Introduction.
2. Case History
3. Determination of ALT
a- Priniciple
b- Reagents
c- Procedures
d- Calculation
4. Case discussion
Transferable skills:
1.Acquire manual skills in handling laboratory equipments
2.Correlate the laboratory findings with theoretical knowledge
READING: Handouts will be distributed before the class
236

Practical 12 : Inborn error of metabolism
Department: Biochemistry
Teaching location: Classroom & Biochemistry Laboratory
By the end of this topic the student shall be able to:
1. Appreciate the concept of screening
2. Utilize screening test for identification of suspected cases of inborn errors
3. Understand the biochemical basis of such diseases
1- Introduction.
2. Case History ( 3 cases)
3. Aminoaciduria
4. Qualitative screening of urine
5. Different tests used in screening:
a-Ferric Chloride test
b- Dinitrophenylhdrazine
c-Cyanide Nitroprusside reaction and test
d- Nitroosonapthol test
e- P- Nitroaniline reaction
6. Cases Discussion
Transferable skills:
1.Acquire manual skills in handling laboratory equipments
2.Correlate the laboratory findings with theoretical knowledge
READING: Handouts will be distributed before the class
237

Practical 13 : Serum Uric Acid
Department: Biochemistry
Teaching location: Classroom & Biochemistry Laboratory
By the end of this topic the student shall be able to:
1. Determine uric acid in serum by spectrophotometric method
2. Correlate the biochemical defect to physical properties of uric acid, to clinical picture of gout
3. Utilize an enzyme inhibitor as a drug
1- Introduction.
2.Case History
3. Blood Uric Determination
a- Priniciple
b- Reagents
c- Procedures
d- Calculation
4. Case discussion
Transferable skills:
1.Acquire manual skills in handling laboratory equipments
2.Correlate the laboratory findings with theoretical knowledge
READING: Handouts will be distributed before the class
238

Practical 14 : Molecular Diagnosis of Genetic Defects (Sickle cell disease)
Department: Biochemistry
Teaching location: Classroom
By the end of this topic the student shall be able to:
1. Understand the basic principles of molecular diagnosis
2. Appreciate the ability of these techniques to diagnose families at risk for sickle cell anaemia
1- Introduction.
2. Case History
3. Genetic diseases
4. Molecular diagnosis of sickle cell anaemia
a- Isolation of DNA
b- Digestion of DNA by restriction enzyme
c- Separation of restriction fragments by electrophoresis
d- Transfer of separated fragments to nitrocellulose (blotting)
5. Polymerase chain reaction (PCR)
6. DNA Preparation from blood in EDTA
Transferable skills:
1.Acquire manual skills in handling laboratory equipments
2.Correlate the laboratory findings with theoretical knowledge
READING: Handouts will be distributed before the class
239

Practical (15) : OSPE TRAINING
Department: Biochemistry
Teaching location: Classroom
By the end of this practical the student will be able to: o Be familiar with the OSPE

8-9 stations will be included, two of them will be related to the previously mentioned tests (determination of the analytes), the other will include questions to evaluate the student benefits of the information given in these practicals.
Transferable skills:

Accuracy of performing the test

Utilization of data in the different stations for interpretation of clinical cases
READING: Laboratory Handouts will be distributed
240