MODULE 2│PHARMBIOSCI 3
BIOCHEMISTRY
BIOCHEMISTRY
•
II. AA with uncharged Polar groups
generally, deals with the physical and chemical properties of
compounds that make up the smallest unit of life and how
these compounds undergo processes and relate it with how
it affects the daily function of human beings.
R–H
PROTEINS
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constitute 70% of the organic matter of cell. The simplest unit
is amino acid, and proteins are polymers of these repeating
units linked together
With OH
Glycine
Gly
G
Serine
Ser
S
Threonine
Thr
T
Tyrosine
Tyr
Y
Asparagine
Asn
N
Glutamine
Gln
Q
Cysteine
Cys
C
AMINO ACIDS
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organic molecule containing both carboxyl and amino
functional groups
•
there are about 300 amino acids occurring in nature, but only
about 20 are commonly occurring and are constituents of
proteins. Except for proline, an imino acid, all 19 are alpha
amino acid with the structure shown below:
With amide
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Sulfur containing
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Cysteine and Methionine
Glycine
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Acts as inhibitory neurotransmitter; antagonized by
strychnine
With SH
CLASSIFICATION OF AMINO ACIDS
I. AA with Non-polar R-groups
Alanine
Ala
A
Valine
Val
V
Leucine
Leu
L
Isoleucine
Ile
I
Proline
Pro
P
Phenylalanine
Phe
F
III. AA with charged Polar groups
Glutamic
Glu
Acid
Alkyl R
group
E
Aspartic Acid
Asp
D
Lysine
Lys
K
Histidine
His
H
Arginine
Arg
R
Alkyl R
group
A. PROTEIN STRUCTURES
•
Aromatic
group
Tryptophan
Trp
W
Structurally organized into four levels (with each level
having its proper specificity): PRIMARY, SECONDARY,
TERTIARY, and QUATERNARY
1. Primary Structure
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S–
containing
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Methionine
Met
M
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* Proline is an alpha imino acid, it doesn’t contain a free amino group
Module 2 – Biochemistry
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The simplest level of structural organization composed of
the amino acid resides linked through peptide bonds
The sequence is written from left to right (N–Terminal to
C–Terminal amino acid
In an electric field Positively charge proteins →Cathode (-)
Negatively charged proteins → Anode (+)
Proteins at the isoelectric Ph → no migration since net
charge is 0
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2. Secondary Structure
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α-Helix
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A helical configuration of the polypeptide chain
formed by the Hydrogen bonding between the
peptide groups of every first and fourth AA residues.
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β-Sheets
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Pleated sheet structure resembling an accordion
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Can be in a form of a parallel or an antiparallel
chain
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Examples are Keratin, Collagen, and Fibroin
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Creutzfeldt – Jakob Disease
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Caused by the transmission of Prions (a
proteinaceous infectious agent that causes
neurodegenerative diseases) that results to the
misfolding of the normal prion protein found
abundantly in the brain
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Prions causes Mad Cow Disease
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Replacement of the normal α-helical
arrangement of the normal prion protein with βpleated sheet
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Dementia and Involuntary Jerking Movements
(Startle Myoclonus)
3.Tertiary Structure
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Refers to the spatial arrangement of the polypeptide chain
Can either be Fibrous or Globular
Different bonds contribute to the stabilization of the
structure
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Covalent bond: Disulfide Bond
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Polar bonds: Hydrogen bonding, Ionic bonding
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Non-Polar/ van der-Waals
Chaperones
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Proteins that assist in the folding and unfolding of the
polypeptide to correctly form the tertiary structure
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Example: Heat Shock Proteins
4. Quaternary Structure
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Spatial arrangement of proteins made up of several
polypeptide chains, with each peptide chain having a
tertiary structure
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Referred to as Oligomers
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Example: Hemoglobin
Denaturation (Denativation) of proteins happen when certain
agents such as heat and urea destroy the higher
Structural levels of protein without destroying the peptide bonds
Ehlers – Danlos Syndrome
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A group of connective tissue disorders that are
generally characterized by hyperextensible skin, joint
hypermobility, and defects in large blood vessels
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3. Insulin
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A polypeptide hormone produced by the beta – cells of the
pancreas
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Synthesis of Preproinsulin in the rough ER → Proinsulin is
cleaved in the secretory granules → C – peptide is released
→ the remaining molecule forms Insulin
C. QUALITATIVE TESTS FOR PROTEINS AND AMINO ACIDS
1. Biuret Test
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Identifies the presence of proteins
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Uses 1% NaOH and a solution of Copper (II) sulfate
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Positive Result: Violet color
2. Ninhydrin Test
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Uses Ninhydrin to detect the presence of amines
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Positive Result: Purple (Ruehlmanns’ Purple) = Amino Acids
with a free amin group, Yellow = Imino Acids (Proline &
Hydroxyproline)
3. Xanthoproteic Test
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For Tyrosine, Tryptophan
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Uses conc. HNO3 and 40% NaOH
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Positive Result: Yellow Color
4. Millon’s test
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Specific for Tyrosine
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Uses Mercury dissolved in conc. HNO3
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Positive Result: Yellow Coloration
5. Hopkin’s – Cole Test
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Specific for Tyrosine (Indole group)
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Uses Glyoxylic Acid in Glacial Acid and Sulfuric Acid
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Positive Result: Purple Color on the surface
6. Nitroprusside Test
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Specific for Cysteine
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Uses nitroprusside in alkaline solution
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Positive Result: Red Coloration
7. Sakaguchi Test
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Specific for Arginine (guanido group)
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Uses NaOH, α-naphthol, and Bromine solution
ENZYMES
Renaturation/(Renativation), on the other hand, is the recovery of
the protein from its denatured state
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B. MEDICALLY IMPORTANT PROTEINS
1. Hemoglobin
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Globular Transport Protein for Oxygen
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Contains Heme which is a complex of porphyrin ring and a
Ferrous
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Sickle Cell Anemia
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Caused by a mutation in hemoglobin where the
Glutamate at position six in the beta–chain of
hemoglobin is replaced by Valine
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Has higher affinity to CO than CO2
2. Collagen
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Found mainly in the bones and cartilage
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Mainly contains Glycine, Proline, Hydroxyproline
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Types:
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I – Found in skin and bones
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Osteogenesis Imperfecta is a disorder in the
synthesis of Type I collagen characterized by a
distinctive blue sclera and predisposed multiple
childhood fracture
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II – Found in the Cartilage
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III – Found in the arterial walls
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IV – found in the basal lamina
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V – Found in the hair and placenta
Module 2 – Biochemistry
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These are biological catalysts of protein nature which
catalyzes energetically feasible reaction without altering the
reaction route. Like other nonbiological catalysts, enzymes
are not consumed during a reaction and decreases the Ea
of a reaction
Enzymes are highly specific for their substrates and
products with most enzymes recognizing only 1 compound
as substrate.
The velocity of a reaction is directly proportional with
temperature
•
However, as the temperature goes higher than 37°,
enzymes start to degrade
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Changes in pH can cause changers in reaction and
may cause enzymes to denature
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Pepsin: optimal pH is 2
Many enzymes require cofactors or coenzymes to catalyzes
reactions. These coenzymes are commonly derived from
vitamins and metal ions.
A. NOMENCLATURE
1. Oxidoreductase
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Catalyzes REDOX reactions
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Also called as dehydrogenases or reductases
2. Transferases
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Catalyzes reactions involving the transfer of different
groups from the substrate to another
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Amino Transferases, Methyl transferases
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3. Hydrolases
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Catalyzes the substrate bond cleavage by adding water
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Amylase, Saccharase
Pyrimidine
4. Lyases
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Catalyze bond – cleaving reactions without oxidation or
addition of water
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Pyruvate decarboxylase
5. Isomerases
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Catalyzes structural rearrangements
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Triose isomerase
6. Ligases
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Also called as synthetases
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Catalyzes the addition of 2 molecules using ATP as energy
source
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DNA – Ligase
B. DNA DOUBLE HELIX
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B. INHIBITION
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Inhibitors are agents capable of exerting a specific deterrent
action on the activity of an enzyme
1. Competitive
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An inhibitor that is structurally related to the substrate binds
to the active center preventing the formation of the enzyme
– substrate complex
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2. Noncompetitive
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An inhibitor binds to the enzymes or enzyme – substrate
complex which leaves the active center free and may
induce conformational hinders the formation of enzyme –
substrate complex.
C. DENATURATION
3. Irreversible
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An inactivator bonds covalently to the enzyme and
inactivate it
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Also known as isozymes
These are enzymes that may differ in amino sequences and
physical properties but catalyze the same reaction.
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A. DNA STRUCTURE
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Upon heating, DNA strands separate the base pairs reform
once the temperature is slowly decreased
E. HYBRIDIZATION
DNA & RNA
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Separation of the DNA strands due to heat or alkali exposure
without breaking the phosphodiester bond
D. RENATURATION
C. ISOENZYMES
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Proposed by Watsons and Crick in 1953
The secondary structure of DNA is formed by the pairing two
polynucleotide chains that are antiparallel.
•
One chain rubs 5’ – 3’ and the other 3’ – 5’
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The base sequences of the two strands are
complementary
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Adenine and Thymine via 2 hydrogen bonds
(major groove)
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Guanine and Cytosine via 3 hydrogen bonds
(minor groove)
One full turn of DNA helix contains nucleotides
Has 3 different conformations:
B form: Right-handed
Z form: Left-handed
A form: dehydrated and compact; Right-handed
A single strand of DNA or RNA pairs with a complementary
base sequence on another strand of DNA or RNA
F. HISTONES
Contains nucleosides that is made up of a nitrogenous base,
deoxyribose, and phosphate
The nitrogenous bases are classified into Purine and
Pyrimidines
The primary structure of DNA and RNA is linear
polynucleotide chain made up of mononucleotides which are
linked by a 3’,5’ phosphodiester bond (3’ C of one sugar to
the 5’ C of the next sugar)
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•
These are small of DNA small, basic proteins rich in Arg and
Lys; not present in prokaryotes
Eukaryotic chromatins consist of a DNA complexed with
histones
G. RNA vs. DNA
The Nucleotides of DNA
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RNA contains ribose as sugar instead of deoxyribose
Uracil replaces Thymine
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Single stranded with extensive base pairing
RNA can sometimes act as catalysts and enzymes
(ribonucleases, Peptidyl transferase)
Purines
H. TYPES OF RNA
1. Messenger (mRNA)
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Contains a cap consisting of a methylated guanine
triphosphate attached to the hydroxyl group on the ribose at
the 5’ end
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The poly(A) tail contains up to 200 Adenine nucleotides
2. Ribosomal (rRNA)
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Aids in the formation of ribosomes
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Prokaryotic: 70s (Large subunit = 50s; Small subunit = 30s)
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Eukaryotic: 80s (Large subunit = 60s; Small subunit = 40s)
Module 2 – Biochemistry
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3. Transfer (tRNA)
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Has a characteristic cloverleaf structure
DNA REPLICATION
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Known as the transfer of genetic formation within single class
of nucleic acids, i.e., from DNA to DNA or, as in certain
viruses from RNA to RNA.
DNA Replication is:
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Bidirectional
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Replication begins at a specific origin and
simultaneously moves out in both directions
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Prokaryotes have one site of origin while
eukaryotes can have multiple sites of origin
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Semiconservative
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The resulting daughter DNA contains one intact
parenteral strand and newly synthesized
complementary strand.
b.
c.
d.
e.
f.
Promoters contain a sequence TATAAT known as the
Pribbenow box or TATA BOX
•
In eukaryotes, Hogness box (TATA Box) has a
sequence of TATATAA
An RNA Polymerase – closed promoter complex is formed
The DNA unwinds at promoter and forms the open promoter
complex
RNA Polymerase initiates the mRNA synthesis with a purine
ribonucleoside triphosphate
RNA Polymerase holoenzyme catalyzes the elongation of
mRNA by about 4 more units
Upon reaching about 10 nucleosides, the sigma – subunit
dissociates and will bind to another RNA Polymerase
2. Termination
a.
Intrinsic (Rho – independent Transcription Termination)
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Controlled by specific sequences called termination
sites wherein the sequences form a hairpin loop
structure that allows the RNA Polymerase to detach
from the DNA template strand
b.
Rho – dependent Transcription Termination
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Involves the Rho – Protein
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The rho –protein binds to the RNA and chases the
polymerase until it reaches the termination site to
facilitate the dissociation of the RNA Polymerase from
the DNA Template Strand.
A. STEPS IN REPLICATION
1. Initiation
a. The parenteral DNA is separated and uncoiled by helicases
where the uncoiled DNA strand serves as the template
b. As the strands separate. An active site for synthesis known
as replication fork is formed where each separated strand is
stabilized by a single – stranded protein
c. As the strands separate, supercoiling happens which is
removed by DNA Topoisomerase
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Etoposide inhibits topoisomerase
d. The primase aids in the production of the RNA primer in a 5’
– 3’ direction
C. EUKARYOTIC TRANSCRIPTION
1. RNA Polymerase (Promoters)
2. Elongation & Termination
a. From the initiation site, DNA Polymerase III adds
deoxyribonucleotides at the 3’ OH end of the primer
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DNA polymerase can only copy as 3’ – 5’ direction and
produce the daughter strand in a 5’ – 3’ direction
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DNA polymerase always requires primers and cannot
initiate the formation of new strands
b. DNA Polymerase III forms continuous strands in a 3’ – 5’
direction forming the leading strand
c. In the 5’ – 3’ strand, a discontinuous formation of DNA
happens forming smaller fragments known as Okazaki
fragments. These fragments are joined together by DNA
ligase
d. Once complete, RNA primer is removed by the exonuclease
activity of DNA Polymerase I
e. Gaps are filled with the complementary bases
f.
Replication is terminated, forming 2 daughter DNA
molecules.
a.
RNA Polymerase I
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Found in the nucleolus and synthesizes the precursors
of ribosomal RNA
b. RNA Polymerase II
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Found in the nucleoplasm and synthesizes mRNA
precursors
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α-Amanitin (a toxin from the mushroom Amanita
phalloides) binds and inhibits RNA Polymerase II which
halts mRNA synthesizes resulting to severe GIT
symptoms, liver toxicity, and death.
c. RNA Polymerase III
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Found in the nucleoplasm and synthesizes the tRNA
precursors
2. Steps in Eukaryotic Transcription
a.
RNA TRANSCRIPTION
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The process of making an RNA form a DNA template is
known as transcription.
Is the major control point in gene expression and protein
production
Catalyzed by DNA – dependent DNA polymerase
No primer is needed
Uses one strand of DNA as template
A. RNA POLYMERASE
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Can initiate synthesis of new chains without primer
Copies the DNA template in a 3’ – 5’ direction and resulting
RNA elongates in a 5’ – 3’ direction
Precursors: Ribonucleoside Triphosphate (ATP, UTP, GTP,
and CTP)
3. Reverse Transcription
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The process of transcribing the single stranded RNA into a
double – stranded DNA
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Enzyme: Reverse Transcriptase (RNA – dependent DNA
Polymerase)
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Retrovirus contain RNA as genetic material
RNA TRANSLATIN AND PROTEIN SYNTHESIS
B. STEPS IN PROKARYOTIC TRANSCRITION
A. GENETIC CODE
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1. Initiation and Elongation
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Rifampin inhibits the beta subunit of bacterial dependent
RNA Polymerase
•
Actinomycin D binds to DNA and inhibits the elongation or
RNA Transcription by RNA Polymerase
a.
mRNA Synthesis
1. RNA Polymerase II produces a heterogenous nuclear
RNA (hnRNA) containing exons and introns
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Exons are sequences appearing in the mature mRNA
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Introns are the non-coding region and are removed
during splicing
2. The primary transcript (hnRNA) is capped at the 5’ end
3. A poly(A) tail with a nucleotide length range of 20 – 200
is added at the 3’ end of the transcript
4. The introns are removed and the exons are connected
to form the mature mRNA through splicing
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A collection of codons that specify all the amino acids found
in proteins
Characteristics:
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It is degenerate (Redundant; Many amino acids have
numerous codons)
•
It is non – overlapping
RNA polymerase is directed by the sigma factor to bind to
the promoter
Module 2 – Biochemistry
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Begins with the start codon AUG (Methionine) near
the 5’ end of the mRNA which determines the
reading frame
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Ends with a stop codon (UGA, UAA, UAG) near
the 3’ end
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The code is comma less (no markers to differentiate
one codon from one another)
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The code is nearly universal
Codons
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A sequence of 3 bases in mRNA that specifies a
particular Amino acid
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g.
3. Termination
a.
b.
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Mutations in DNA are carried over into the mRNA that
causes changes in the encoded protein
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1. Point mutations
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A base in the DNA is replaced by another which alters the
codon in the MRNA
a.
b.
The stop codon is recognized by a release factor
The newly synthesized protein is released and the
synthesizing complex dissociates
CARBOHYDRATES
B. MUTATIONS
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The steps are repeated until the ribosome encounters a stop
codon
Silent
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The codon containing the changed base codes for
the same amino acid
Missense
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The codon containing the changed base codes for a
different amino acid
The most widespread compounds involved in the buildup
functions of the cell
Presence of a carbonyl (Aldo or Keto group) and at least two
hydroxyl groups
Stereoisomers: Same chemical formula but differs in the
position of hydroxyl groups
Enantiomers: Stereoisomers that are mirror images
Epimers: Stereoisomers that differ in the position of the
hydroxyl group at only one asymmetric carbon.
A. MONOSACCHARIDES
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Nonsense
•
The codon containing the changed base codes for a
stop codon
2. Insertions
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Occur when a base or a number of bases are added to the
DNA
These are simple carbohydrates that are named depending
on the number of Carbon atoms and the specific carbonyl
group present
They can either be Dextrorotatory or Levorotatory
Examples: D – Galactose, L - Fructose
c.
B. OLIGOSACCHARIDES
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3. Deletions
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Occur when a base or a number of bases are deleted to the
DNA
4. Frameshift
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Occurs when the number of bases added or deleted is not a
multiple of 3 which shifts the reading frame to a completely
different set of codons
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C. STEPS IN EUKARYOTIC TRANSLATION
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C. POLYSACCHARIDES
1. Initiation
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a.
b.
c.
The 40s ribosomal subunit binds close to the 5’ cap until it
recognizes the start codon (AUG).
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The ribosomes recognize the AUG in the correct
context known as the Kozak Sequence (ACCAUGG)
•
In prokaryotes, Streptomycin binds to the 30s subunit
which hinders initiation
A special initiator tRNA recognizes the start codon carriers a
non formylated Methionine
The 60s ribosomal subunit is recruited from the 80s initiation
complex.
c.
d.
e.
f.
The ribosome moves from 5’ to 3’ direction
The amino acid containing tRNA (aminoacyl-tRNA’s) forms a
complex with elongation factor and enters the empty A-site
The anticodon of the aminoacyl-tRNA is matched against the
mRNA codon in the A-site
When the correct aminoacyl-tRNA enters the A-site, the
polypeptide chain in the P-site is linked to the new amino
acid in the A-site via a peptide bond
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The reaction is catalyzed by peptidyl transferase
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Chloramphenicol inhibits prokaryotic peptidyl
transferase
•
The new peptidyl-tRNA is left in the A site
The peptidyl-tRNA is translocated from the A-site to the Psite facilitated by an elongation factor
•
Erythromycin binds to a site on the 50s subunit which
inhibits translocation
A new aminoacyl-tRNA in the A-Site is made available for
the next codon in the mRNA
Module 2 – Biochemistry
These are high – molecular carbohydrates with more than
ten monosaccharide units linked by glycosidic bonds
Neutral Polysaccharides such as Starch (Plants) and
Glycogen (human and animals) are found inside the cells as
reserve material
Acidic polysaccharides such as Chondroitin sulfate and
Hyaluronic acid are found extracellularly
D. DIGESTION OF CARBOHYDRATES
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2. Elongation
a.
b.
Composed of 2 to 12 monosaccharide units linked by
glycosidic bond
Sucrose
Table Sugar
Glucose + Fructose
Lactose
Milk Sugar
Galactose – linked β-1,4 to glucose
Salivary α-amylase, which is present in the saliva, cleaves
starch by breaking the α-1,4 linkage between glucose
residues
Sucrase converts sucrose to glucose and fructose
Lactase coverts lactose to glucose and galactose
α-glucosidase inhibitors work in the intestine to slow down
the digestion of carbohydrates to facilitate better post
digestive blood glucose control
E. QUALITATIVE TESTS FOR CARBOHYDRATES
1. Molisch Test
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General test for Carbohydrates
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Monosaccharides give the most rapid result
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Uses α-naphthol and sulfuric acid
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(+) result: purple ring
2. Benedict’s test
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Tests for the presence of reducing sugars
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All monosaccharides give a positive result
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Uses Copper (III) sulfate, Sodium citrate and Sodium
carbonate in a mildly basic solution
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(+) result: red to orange precipitate
3. Barfoed’s Test
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Uses Copper (III) in a slightly acidic medium
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Used to differentiate a reducing monosaccharide from a
reducing disaccharide
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(+) result: red ppt (within 3 minutes = Monosaccharide,
longer than 3 minutes = disaccharide)
Anabolism
External
trigger
4. Bial’s Test
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Test for differentiating pentose and hexose monosaccharides
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Used concentration HCl and orcinol + ferric chloride
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(+) result: Pentoses = bluish to green, Hexoses + brownish
to gray
Endocrine
gland
Negative
feedback
Hormone
5. Seliwanoff’s Test
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Test for differentiating aldoses from ketoses
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Uses 6M HCl and resorcinol
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(+) result: Ketoses = Cherry red, Aldoses = bluish-green to
peach
Target
organ
TOPICS ON HUMAN PLANT AND METABOLISM
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INTRODUCTION
What is metabolism?
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Metabolism is the sum of all chemical reactions in the body
(the reactions are catalyzed by specific enzymes)
A series of metabolic steps with a specified end-product is
called a metabolic pathway
•
Metabolic pathways are linear or cyclic
•
Metabolic pathways are either catabolic or anabolic
•
Each pathway usually has an irreversible reaction to
dictate the direction of the process
A↔B↔C→D
Next question: How do we know if the catabolic or
anabolic process will dominate?
•
Answer: control the irreversible steps (often by negative
feedback). When one process is on, the opposite must be
off
Why do we need metabolism?
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To generate energy
Humans are thermodynamically open systems, and the second
law states that entropy (disorder) must increase.
Humans that don’t produce energy will give up to entropy, their
bodies will lose homeostasis, and they die.
Entropy is counteracted it performs. We don’t die as result
ATP will serve as the primary energy currency.
Other cofactors can also be
equated to ATP:
•
NADH (reduced
NAD+) = 2.5 ATP
•
FADH (reduced
FAD+) = 1.5 ATP
•
Other triphosphates
(ex. GTP) = 1 ATP
*NADH and FADH2
will only become
ATP after going
through the electron
transport chain
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“______lysis” “______oxidation”
Catabolism
Smaller
molecules
Larger
molecules
OVERVIEW
Energy
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•
Anabolism
Respiration involves most biomolecules
They converge at acetyl-CoA, which ultimately enters the
citric cycle
Carbohydrate
Protein
Fat
“______genesis” “______synthesis”
Digestion and absorption
Catabolism – breaking down
Anabolism – building up
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Catabolism and anabolism, by direction, are opposites
Do you think the same exact enzymes can perform the
opposite processes?
Answer: NO Different enzymes must be used because
each pathway has at least one irreversible step.
If there is no irreversible step, a “futile cycle” will
result (pathways moving around without direction and
achieving totally nothing)
Catabolism
Simple sugars
(mainly glucose)
Fatty acids
+ glycerol
Amino acids
Catabolism
Smaller
molecules
Acetyl-CoA
Larger
molecules
Energy
ETC
Module 2 – Biochemistry
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INTRODUCTION
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Cell respiration and carbohydrate metabolism are not one
and the same!
Yet, they are discussed together because carbohydrates are
commonly used materials in respiration
Familiar processes:
•
Glycolysis
•
Krebs’ Cycle
•
Electron Transport Chain
Classification equation for cell respiration
C6H12O6 + 6O2 → 6CO2 + 6H2O + ATP
Complete set of processes:
•
Glycolysis and gluconeogenesis
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Glycogen metabolism (glycogenesis and glycogenolysis)
•
Pentose phosphate pathway
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Metabolic effects of insulin and glucagon
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Krebs’ Cycle
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Electron Transport Chain
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CARBOHYDRATE METABOLISM
•
Digestion
first!
Carbohydrates from
food intake
DIGESTION
Pentose phosphate
pathway
GLYCOGENOLYSIS
Glycogen
Glyceraldehyde-3phosphate (GAP)
will proceed to
payoff
Dihydroxyacetone
phosphate
(DHAP) will not
poceed)
For DHAP to
proceed it must
isomerize into
GAP (on top of
the original
GAP)
Gives a total of
TWO GAP
molecules →
everything in
the payoff
phase must
be multiplied
by TWO!
Pentoses
+
CO2
Glucose
↑↓
Glucose 6-phosphate
GLYCOGENESIS
GLUCONEOGENESIS
GLYCOLYSIS
Pyruvate
Lactate
1. GLYCOLYSIS
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aka Embden-Meyerof-Parnas pathway
breakdown of glucose to two molecules of pyruvate
Cytosolic
Energy-producing
Consists of two phases
•
Energy investment (steps 1-5)
•
Energy payoff (steps 6-10)
ATP yield?
•
2 ATP produced (7 ATP if NADH is processed)
Effect on blood sugar?
•
Lowers blood sugar
•
Works during fed states (after eating)
•
Stimulated by insulin
Irreversible steps?
•
3 steps: #1, #3, & #10
“The committed step”?
•
Rate-limiting step – step 3
Module 2 – Biochemistry
Page 7 of 19
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Pyruvate
•
end product of glycolysis can be converted back to glucose.
Glucose
Synthesis of
glycogen
Glucose-6phosphate
Glycogen
Pentose phosphate
pathway
Ribose,
NADPH
Degradation of
glycogen
Glycolysis
Gluconeogenesis
Pyruvate
•
G6P bridges many pathways in carb metabolism
•
Just because G6P cannot go back to glucose, it doesn’t
mean it fully commits to glycolysis
•
Step 1 can NOT be the committed step
The next irreversible step (Step 3) is
•
The enzyme for step 3, PFX, is highly regulated
•
2. FATES OF PYRUVATE
Pyruvate
Aerobic conditions in
humans, animals,
and microorganisms
ACETYL CoA
Anaerobic conditions
in humans, animals,
and microorganisms
Anaerobic conditions
in some
microorganisms
LACTATE
ETHANOL
NEW ENZYMES (only 4):
•
•
•
To reverse step 10:
•
Pyruvate carboxylase*
•
PEP carboxykinase
To reverse step 3:
•
Fructose-1,6-biphosphatase
To reverse step 1:
•
Glucose-6-phosphatase (G6Pase)
•
G6Pase is only in the liver
*Do not confuse with pyruvate decarboxylase!
liver – 90%
kidney – minor; more likely 10%
Amino Acids, Glycerol
•
•
Amino acids – through the citric acid cycle
Glycerol – via the following reaction
3. GLUCONEOGENESIS (GNG)
•
•
•
•
•
Conversion of non-carbohydrates into glucose
Substrates include pyruvate, amino acids, glycerol, and
lactate
Similar to reverse of glycolysis
•
Irreversible steps require different enzymes
Timing: fasted state (which release of glucagon)
Effect: increased blood glucose
Module 2 – Biochemistry
Page 8 of 19
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Uridine diphosphate glucose (UDPGlc).
I. GLYCOGENESIS
•
•
•
•
Lactate
•
•
•
To reverse step 1:
•
Glucose-6-phosphatase (G6Pase)
•
G6Pase is only in the liver
•
Thus, ~90% of GNG occurs in the liver
The GNG of lactate is cyclic.
•
Cori Cycle
•
Synthesis of Glycogen
Anabolic
Requires formation of Contains α1, 4 and α1,6 bonds
Stimulated by:
•
Insulin
Effect:
•
reduce blood glucose
Timing:
•
Fed state
The biosynthesis of glycogen. The mechanism of branching as
revealed by feeding 14C-labeled glucose and examining liver
glycogen at intervals
Steps:
1.
2.
3.
4. FATES OF GLUCOSE-6-PHOSPHATE
A. GLYCOGEN METABOLISM
Glycogen
•
A branched homopolysaccharide
•
Contains α1, 4 (linearity) and α1,6 (branching) glycosidic
bonds
G6P to G1P by phosphoglucomutase (PGM)
G1P + UTP → UDP-glucose
UDP-glucose is added to a glycogen molecule (Glcn) by
Glycogen synthase
•
After step 3, glycogen becomes longer (Glcn+1)
•
The branching enzyme uses part of the chain to
make α1,6 bonds
II. GLYCOGENOLYSIS (GGL)
•
•
•
•
•
•
Breakdown of glycogen
Catabolic
Requires hydrolysis of α1, 4 and α1,6 bonds
Stimulated by:
•
Glucagon
Effect:
•
Increase blood glucose
Timing:
•
Fasted state
Steps:
1.
Glycogen phosphorylase cleaves a glucose molecule from
glycogen into G1P
2. G1P → G6P by PGM
3. G6P → glucose by G6Pase
•
The glucose goes to the blood and to the organs that
need it
•
Any branches block phosphorylase, and must be removed by
the debranching enzyme
III. GLYCOGEN STORAGE DISEASES
•
•
Diseases in the metabolism of glycogen
Commonly lead to hypoglycemia, hyperlipidemia, and/ or
hepatomegaly; some are fatal.
Pathways of glycogenesis and of glycogenolysis in the liver.
, Stimulation;
Module 2 – Biochemistry
, inhibition.).
Page 9 of 19
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GSD TYPE (common name)
0
1 (Von Gierke’s)
2 (Pompe’s)
3 (Cori’s)
4 (Andersen’s)
5 (McArdle’s)
6 (Hers’)
7 (Tarui’s)
ENZYME DEFICIENT
Glycogen synthase
G6Pase
Lysosomal alpha-glucosidase
Debranching enzyme
Branching enzyme
Muscle phosphorylase
Liver phosphorylase
PFK
B. PENTOSE PHOSPHATE PATHWAY
•
•
•
•
*All reactions are irreversible
*Step 1 is the major rate-limiting step
Oxidative phase is controlled by level of NADP+
NADPH is not NADH!
Uses of NADPH:
•
Lipid biosynthesis
•
Detoxification (in conjugation with glutathione (GSH))
THE THING CALLED GLUTATHIONE
•
aka HEXOSE MONOPHOSPHATE SHUNT)
Another pathway for G6P
An alternate pathway of glycolysis in the cytosol
Consists of two phases
I.
Oxidative
II. Non-oxidative
•
•
Essential for protection of the cell against oxidative and
chemical insults
GSH is oxidized to the disulfide form by help of glutathione
peroxidase to counteract oxidative stress
Most of the NADPH in the RBC is used by glutathione
reductase to maintain GSH in the reduced state
•
G6PD is a key enzyme in the conversion of NADP to NADPH
(and therefore, is also key in glutathione activity)
•
Imagine the sequence of events that follow if there is G6PD
deficiency
*G6PD is deficient if there is minimal amount of NADPH, there is
also minimal amount of active glutathione. If there is minimal amount
of active glutathione, the we will not be able to detoxify Hydrogen
peroxide which leads to ↑ = damage → cell death; RBCs: hemolysis
•
•
In G6PD-deficient people, their cells (especially RBC) are
susceptible to oxidative stress
When triggered by drugs or reactive oxygen species (ROS),
causes acute hemolysis (and related consequences jaundice
and dark urine)
II. NON-OXIDATIVE PHASE
•
•
Leads to synthesis of other sugar phosphates
Merely a shuffling of carbons between pairs of sugar
I. OXIDATIVE PHASE
•
•
•
Involves three steps
Riboluse-5-phosphate serves as the final product
Steps 1 and 3 produce NADPH
*All reactions are reversible
*Non-oxidative state phase is controlled by the requirements of
pentoses
•
•
Module 2 – Biochemistry
Page 10 of 19
Can simply lead back to glycolysis or lead to Ribose-5Phosphate for nucleotide synthesis
PPP and glycolysis work simultaneously
RJAV 2022
•
•
BUT any excess demand for R5P or NADPH puts priority on
PPP over glycolysis
Otherwise, glycolysis is usually dominant
Series of electron transfers that generates a proton gradient to
fuel the synthesis of ATP
“Series of electron transfers…”
5. CITRIC ACID CYCLE
•
•
•
•
aka Krebs cycle*/ tricarboxylic acid (TCA) cycle
Cycle that converts acetyl CoA to two molecules of CO2
The “central hub”
Takes place at the mitochondrial matrix
Acetyl-CoA → 2CO2
*There is another Krebs cycle, in fact, the citric acid cycle is the
second cycle that Hans Krebs discovered!
•
•
There is a certain flow, and each electron acceptor is
immediately followed by another
Oxygen is the last electron acceptor
•
In fact, ~90% of oxygen we breathe is used in the ETC
•
(the remaining oxygen is used by WBC to fend off
pathogens)
“that generates a proton gradient…”
•
•
•
•
3 NADH
1 FADH2
1 GTP
The Citric Acid Cycle acid is amphibolic: (Catabolic or
Anabolic)
•
Citrate – fatty acid synthesis
•
α-kg – amino acid catabolism
•
Succinyl-CoA – heme synthesis
•
OAA – GNG and nucleotide synthesis
Depends on balance of body acetyl-CoA and OAA levels (to
be discussed more in lipid metabolism)
•
•
“…to fuel the synthesis of ATP.”
Complex I: NADH-CoQ reductase
Complex II: Succinate-CoQ reductase
Complex III: CoQ-cytochrome c reductase
Complex IV: Cytochrome c oxidase
•
•
Oxidative Phosphorylation
•
•
NADH and FADH2 are oxidized to NAD+ and FAD, which
allows the ETC to happen in the first place (and ultimately
result to ATP synthesis)
Very different from other types of phosphorylation
•
Substrate-level phosphorylation
•
Photophosphorylation (in photosynthesis)
But they can get flux back to the matrix using complex V
Complex V actually uses proton influx to create ATP
•
Proton gradient generation and ATP synthesis are
different events, but they are coupled by complex V
(chemiosmotic coupling)
Coupling them is their way of being regulated:
•
•
•
•
•
Module 2 – Biochemistry
When the protons leave the matrix into the IMS, they can’t
get back using any complex from I to IV
Page 11 of 19
The ETC will work only when ATP levels are low, and will
produce ATP as a response
The ETC will slow down when ATP levels are high
Some cells in the body have two capacities to disconnect the
two processes using uncouplers (ex. thermogenin, 2,4-DNP)
•
The ETC (and proton gradient) goes on without creating
ATP
•
If the ETC goes on with creating ATP, the energy
resulting from the process manifests as heat
•
Serves as heat-generating mechanism for some
animals
All complexes must form a continuous flow
Blocking the complexes can lead to severe complications!
RJAV 2022
We need some computations, too
LIPID METABOLISM
Lipids with Fatty Acids (Saponifiable)
•
TAGs
•
Phospholipids
Lipids without Fatty Acids (Non-saponifiable)
•
Terpenes
•
Sterols
SUMMARY ON CELL RESPIRATION (with ATP count)
1. LIPOGENESIS
Citric Acid Cycle
•
FATTY ACID SYNTHESIS
Energy yield per acetyl-CoA:
OLD CONVERSION
(NADH = 3 ATP │ FADH =
2 ATP)
3 NADH
1 FADH2
1 GTP
TOTAL
CURRENT
CONVERSION
(NADH = 2.5 ATP │ FADH =
1.5 ATP)
9
2
1
12 ATP/ acetyl
2.5
1.5
1
10 ATP/ acetyl
•
•
Modern ATP Computation (aerobic respiration)
PROCESS
Glycolysis
2 pyruvate to 2
acetyl
2 x acetyl to 2
CO2 (TCA
cycle)
•
MOLECULES PRODUCED
2 ATP
2 NADH x 2.5 = 5 ATP
Minus two if GP shuttle is
used
2 NADH x 2.5 = 5 ATP
ATP YIELD
5–7
3 NADH x 2.5 = 7.5 ATP
1 FADH x 1.5 = 1.5 ATP
1 GTP x 1 = 1 ATP
Total of 10 ATP/ acetyl
20
•
Occurs in the cytosol
RLS: Conversion of acetyl-CoA to malonyl-CoA by acetylCoA carboxylase (ACC)
When synthesized, fatty acids are esterified into
phospholipids and triglycerides
Timing: Fed state (stimulated by insulin)
5
30 – 32
Overall
Old ATP Computation (aerobic respiration)
PROCESS
Glycolysis
2 pyruvate to 2
acetyl
2 x acetyl to 2
CO2 (TCA
cycle)
MOLECULES PRODUCED
2 ATP
2 NADH x 3 = 6 ATP
Minus two if GP shuttle is
used
2 NADH x 3 = 6 ATP
ATP YIELD
6–8
3 NADH x 3 = 9 ATP
1 FADH x 2 = 2 ATP
1 GTP x 1 = 1 ATP
Total of 12 ATP/ acetyl
24
Overall
Module 2 – Biochemistry
6
36 – 38
Page 12 of 19
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2. LIPOLYSIS
A. DIGESTION AND GENERAL LIPOLYSIS
Digestion in GIT:
•
A small number of TAGS are digested by lipases (can be
found in intestines)
•
A larger amount is converted into micelles by bile acids –
emulsification → further absorption
Breakdown in fat:
•
Triggered by the fasted state, with glucagon release or
insulin inhibition
•
Initial hydrolysis of triglycerides by hormone-sensitive
lipase (HSL) into glycerol and fatty acids.
How about Phospholipids?
•
•
•
•
•
•
B. FATTY ACID OXIDATION
aka beta-oxidation
Nearly complete reverse of fatty acid synthesis
Timing: Fasted state (stimulated by glucagon)
Successive breakdown of fatty acids by 2 carbons at a time
(acetyl-CoA)
•
All acetyl-CoA will be oxidized in the TCA cycle
Occurs in the mitochondrial matrix
Requires transport of fatty acids to the matrix!
“Nearly complete reverse of fatty acid synthesis”
Module 2 – Biochemistry
Page 13 of 19
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What is the next ATP production for the complete oxidation of lauric
acid, C12 saturated fatty acid, to CO2 and H2O?
Cycles: 5 x 4 ATP =
Acetyls: 6 x 10 ATP =
“Successive breakdown of fatty acids by 2 carbons at a time”
20
60
(-2)
= 78
Shortcuts:
#ac = #C / 2
#cy = #ac – 1
3. FATES OF HMG-CoA (hydroxymethyl glutaryl – CoA)
3x acetyl-CoA
“Requires transport of fatty acids to the matrix”
A. MEVALONATE PATHWAY
•
•
•
•
•
•
Module 2 – Biochemistry
Page 14 of 19
Pathway for cytoplasmic synthesis of sterols and terpenes*
Stimulated in the fed state (by insulin)
Building block: acetyl-CoA
*In non-animals like plants
Most are also under terpenes
Terpenes are made up of isoprene units (right)
Gives rise to many odors given off by plants
RJAV 2022
VOLATILE OILS
•
Most are also under terpenes
•
Give rise to many odors given off by plants
•
Isoprene 5C
TYPE
Monoterpene
Diterpene
Sesquiterpene
Triterpene
Tetraterpene
# ISOPRENES
2
4
3
6
8
# CARBON
10
20
15
30
40
Sunlight + Liver + Kidney = Good bones
B. KETOGENESIS
•
•
•
•
•
*Happens in cytosol
Production of ketone bodies in the mitochondrion
•
Acetone
•
Acetoacetate
•
Beta-hydroxybutyrate
Timing: Fasting/ Starvation
•
Accompanies β-oxidation due to acetyl-CoA overflow
•
TCA cycle saturates → excess acetyl-CoA become
HMG-CoA, and then acted upon by HMG-CoA lyase
Our tissues normally shift from using sugars to using fats in
the fasted state
The brain cannot use the fats due to the blood-brain barrier
Ketone bodies serve as the emergency fuel of the brain in
these cases
High blood Glucose
(after meal)
Low blood Glucose
(between meals)
Liver
Adipose
tissue
Module 2 – Biochemistry
Page 15 of 19
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•
Skeletal
Muscle
•
•
Ketoacidosis – excessively elevated ketone bodies,
causing acidification of the blood
Ketogenic diet – weight loss; induced Ketosis (but was used
for what before?) ↑fats, ↓carbs, regular protein
Diabetic Ketoacidosis
Heart
NUCLEOTIDE METABOLISM
•
•
Brain
Purine and pyrimidine nucleotides are synthesized
separately
Often synthesized using de novo (“of new” or “from scratch”)
pathways or salvage (from readily-made nucleotides)
I) PYRIMIDINE METABOLISM
SYNTHESIS AND UTILIZATION OF KB’s
•
OMP (Orotidine monophosphate) – parent pyrimidine
FOLATE
II) PURINE METABOLISM
•
Ketosis – shift from dependence on glucose to dependence
on ketone bodies
•
Ketonemia – elevated KB in blood (20mg/100mL)
•
Ketonuria – elevated KB in urine (70mg/100mL)
Module 2 – Biochemistry
Page 16 of 19
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4. AMINO ACID DERIVATIVES
•
Amino acids can be used as precursor for many different
compounds, including:
•
Neurotransmitters
•
Hormones/ Autacoids
•
Pigments
•
Cofactors/ Carriers
•
Secondary plant metabolites (alkaloids, flavonoids,
tannins)
A. FROM TYROSINE
•
•
•
•
Catecholamines
•
DA, NE, E
•
Adrenergic NTs
Thyroid hormones
•
T3, T4
Melanin – enzyme Tyrosinase
•
Primary skin pigment
Phenylpropanoids*
D. FROM ARGININE
•
•
Nitric oxide (NO)
•
Vasodilatory substance
•
Related to MoA of some direct vasodilators
Urea
•
Final product of protein catabolism
E. FROM SERINE
•
•
•
Sphingosine
•
Formed by condensation of serine with palmitic acid
Ethanolamine
•
Found in phosphatidylethanolamine
Choline
•
Found in phosphatidylcholine/ acetylcholine
B. FROM TRYPTOPHAN
•
•
Serotonin
•
Autacoids and Neurotransmitters
Melatonin
•
Sleep-wake cycle
F. MISCELLANEOUS
•
•
•
S-adenosylmethionine (SAM)
•
From methionine
•
One-carbon transport
GABA
•
From glutamic acid
L-carnitine
•
From lysine
•
Tranexamic acid
C. FROM HISTIDINE
•
•
Histamine
•
Contributes to allergic reactions
•
•
Module 2 – Biochemistry
Page 17 of 19
6-aminopenicillanic acid
•
From cysteine and valine
Glutathione
•
Tripeptide of E, C and G
•
Endogenous antioxidant
REMEMBER: all pituitary and hypothalamic hormones are
also peptides!
RJAV 2022
G. PORPHYRIN METABOLISM
•
•
•
•
•
•
•
Porphyrins – chelating macrocycles
•
Starts from the condensation of glycine and succinylCoA
•
Includes heme and chlorophyll
Heme:
•
Heme is incorporated to RBCs as hemoglobin
•
When the RBCs die, the hemoglobin releases heme
and is metabolized in the liver to waste product bilirubin
•
Hyperbilirubinemia can lead to jaundice
•
Other derivatives include stercobilin (feces) and
urobilin (urine)
Carbon monoxide (CO) made from biliverdin production is
toxic (it competes with oxygen in hemoglobin binding)
Conversion of heme all the way to bilirubin happens in the
spleen, but after, is delivered to the liver.
3 MAJOR PROCESSES:
A. Transamination
B. Oxidative deamination
C. Urea cycle
A. AMINO ACID CATABOLISM: TRANSAMINATION
•
•
•
•
•
Glutamate collects amino groups in most tissues and is
converted to glutamine
Pyruvate collects amino groups in muscles and is converted
to alanine
Alanine goes to the liver and is converted back to pyruvate,
but aKg is converted to glutamate
The liver therefore collects all amino groups for further
processing
All above processes are transamination processes, where
pyridoxal phosphate (PLP) is the main cofactor
Bilirubin has to be glucuronidated first before being ready for
excretion
Treatment for neonatal jaundice: Phototherapy
Before: Phenobarbital (enzyme inducer)
5. AMINO ACID CATABOLISM
•
•
•
•
Excess amino acids are not stored
Nitrogen balance
•
Positive: intake > output
•
Negative: outtake > input
Metabolized to ammonia by different tissues in the body
Converted to urea in the liver
•
Urea is the final form
B. AMINO ACID CATABOLISM: [O] DEAMINATION
•
•
In the liver, deamination of glutamate will lead to release of
ammonia (ionized: ammonium)
Deamination is accompanied b replacement of a keto group
→ “oxidative deamination”
C. THE UREA CYCLE
•
•
•
•
Module 2 – Biochemistry
Page 18 of 19
Aka: Krebs-Henseleit cycle
Ammonia is so far the product from deamination
Ammonia is toxic to the body
Ex. when it accumulates in the brain, it can result to hepatic
encephalopathy
RJAV 2022
•
•
Therefore, ammonia in the liver is further converted to urea
(final product of nitrogen disposal)
Involves arginine and nonstandard amino acids citrulline
and ornithine
Name
AA Affected
Alkaptonuria
Tyrosine
Albinism
Tyrosine
Homocystinuria
Methionine
Phenylketonuria
(PKU)
Maple syrup
urine disease
(MSUD)
Phenylalanine
Branched-chain
amino acids
Enzyme
Deficient
Homogentisate
oxidase
Tyrosinase
Cystathionine
synthetase
Phenylalanine
hydroxylase
Branched-chain
keto acid
dehydrogenase
Effects
Crippling
arthritis
Light
complexion
photophobia
Ectopia lentis,
osteoporosis
Retardation,
ketonuria, diet
restrictions
COMMON INBORN ERRORS OF METABOLISM (IEM)/
AMINOACIDOPATHIES
Module 2 – Biochemistry
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RJAV 2022