Determination of inhibition kinetics
Fig 2..2 Effects of inhibitors on Lineweaver-Burk and Eadie-Hofstee plots
Problems
1. An enzymatic assay was carried out using a pure substrate S. The assay was then repeated with
addition of an inhibitor. The results are tabulated below.
[S]/
Vo
10-5 M
No inhibitor
with
inhibitor
1.5
0.21
0.08
2.0
0.25
0.1
3.0
0.28
0.12
4.0
0.33
0.13
8.0
0.44
0.16
16.0
0.40
0.18
a. Plot the data using both Lineweaver-Burke and Eadie-Hofstee Plots
b. Calculate the values of Vmax and Km from both plots
c. Comment on the differences between the two plots
d. Comment on the effect of the inhibitor on the reaction.
2. The action of an enzyme on two different substrates was investigated with the results below.
[S]
V0
/10-5 M
S1
S2
1.67
6.05
5.0
2.50
8.20
6.6
3.33
9.98
8.0
5.00
12.86
10.0
7.00
23.00
11.7
10.00
28.00
13.3
15.00
32.60
15.0
16.70
33.80
15.4
20.00
36.00
16.0
a. Plot the data using both Lineweaver-Burke and Eadie-Hofstee Plots.
b. Comment on the results as shown by these two plots.
c. Calculate the values of Vmax and Km in each case.
IUG , Faculty of Science
Dept. of Chemistry and Biochemistry, Second Midterm Exam
Metabolism (Biochemistry B 2301), 27/12/2014
Answer the Following Questions
First: Account for the following
1- Absence of Glucose-6phosphatase in muscle cells.
2- PFK1 is inhibited feedback by ATP.
3- Krebs cycle is an aerobic process
4- Biosynthesis and degrative pathways are almost distict.
5- Glycolysis pathway is either aerobic or anaerobic process
Second: With structral formula, write the following
1-Coversion of Pyruvate to lactate
2-A Second half of Glycolytic reactions
3-Mechanism of action of Alfa-ketoglutarate dehydrogenase complex
Fourth: Without structral formula, write the following
1-Fractose conversion to glucose.
2-Nonoxidative reactions of Krebs cycle
3-Acetyl CoA conversion to glucose
Fifth: Write the net equation of the following:
1- Complete oxidation of Glucose (state # of ATP produced and calculate effeciency).
2- Gluconeogenesis
IUG , Faculty of Science,
Dept. of Biochemistry
Enzymes, 1st Semester,
Mid-term Exam, 3/12/2014
Answer the Following Questions
I)
Give reason(s)
1) V vs [E]T of single substrate enzyme catalysed reaction is linear.
2) Free energy value of a biochemical rxn is not affected by precence of the enzyme catalyst
3) HK is more important enzyme than GK.
4) C.E substitutes Kcat for studying enzyme effeciency
5) Organophosphorous cpds used for treatment of many plant diseases.
II)
Explain
1) Give an example about control of an enzyme catalyzed reaction by covalent modification
and another about allosteric effector.
2) Three evidences about induce fit mechanism
3) Design an experiment about effect of pH on enzyme activity.
III)
Integrate M. M. equation and how is the final equation used to determine Vmax and
Km
IV) From the reciprocal plot of the following enzyme catalyzed data determine: Km, Vmax.
[S]/ 10-5 M V0 nM/min
1.5
0.21
2.0
0.25
3.0
0.28
4.0
0.33
8.0
0.44
16.0
0.40
a) At initial substrat concentration of 4.0x10-5 M, what is Fes?
b) If total enzyme concentration is 5 microgram, what is the C.E.?
IUG , Faculty of Science,
Dept. of Biochemistry
Enzymes, 1st Semester,
nd
2 Mid-term Exam, 20/12/2014
Answer the Following Questions
I)
1)
2)
3)
4)
Give reason(s)
V vs [E]T of single substrate enzyme catalysed reaction is sometimes hyperbolic.
Rate of a biochemical rxn is speeded up by enzyme catalyst
Substrate in vivo = or less than Km.
Human body is affected by organophosphours cpds.
5) Explain
1) Three pionts attachment theory with one example
2) Design an experiment about effect of substrate on enzyme activity.
1
Starting from M. M. equation, prove that Ln [s]0/[s] = Kt for an enzyme
catalysed reaction and show the plot.
IV) From the reciprocal plot of the following enzyme catalyzed data determine: Km, Vmax.
[S]x 10-5 M V0 nM/min
1.5
0.21
2.0
0.25
3.0
0.28
4.0
0.33
8.0
0.44
16.0
0.40
c) At initial substrat concentration of 5.0x10-5 M, what is Fes? What is its meaning?
d) If total enzyme concentration is 5.0 Micromole/ml, what is Kcat, 1/Kcat, and
C.E.? what are their meaning?.
Dept. of Biochemistry
Final Exam, Enzymes, 1st Semester, 11/1/2014
Answer the Following Questions
(I)
Define: Kcat, isoenzymes, E.U, Fes, purification factor, % recovery (yield)
(II) Give reasons
1- V vs pH for enzymes catalysed reaction in wide range pH is not linear.
2- E.U/ml is used for quantitation of enzyme activity.
3- Tyrpsin cleaves a polypeptide beyond Lys or Arg.
4- Alf-ketoglutarate is present in the body in complex form
5- Kcat is not suitable for studying enzyme catalyzed reactions in vivo.
(III)
Explain
1- Importance of the reciprol plot
2- Importance of Vmax detrmination
3- Steps in enzyme purification
IV) Derive M. M. equation of substrate inhibition by rapid equilibrium of Enz-catalyzed rxn
(1) Rearrange it to give 1/V as a function of 1/[S] and plot it.
(2) Sketch the same plot in presence of CI, UCI and NCI.
V) Salicylate inhibits the catalytic action of Glu dehydrogenase as shown in the following data.
Substrate (mM)
micromole/min (-I )
micromole/min (+I)
1.5
0.21
0.08
2.0
0.25
0.10
3.0
0.28
0.12
4.0
0.33
0.13
8.0
0.44
0.16
16.0
0.40
0.18
1) Determine type of inhibition. For control and inhibitor, find Vmax , and Km.
2) Assume the concentration of salicylate is held constant (40 mM), calculate Ki.
3) If total enzyme concentration is 5.0 Micromole/ml, what are C.E.and their meaning
for control and inhibitor?.
(VI) A pure enzyme preparation produced 0.03 mmoles product in 15 min. under standard condition.
(1) Calculate Vmax as E.U. and S.A. of 3 mg enzyme preparation.
(2) Calculate Kcat and 1/Kcat and explain their meaning (M.Wt is 65000 Gm/mole)
IUG , Faculty of Science, Dept. of Chemistry and Biochemistry,
Metabolism (Biochemistry B 2301), 19/1/2015, Final Exam
Answer the Following Questions
I- Give reasons (4 marks) (1) Biosynthesis and degradation are almost distinct. (2)
Glycogen is a branched molecule. (3) TAGs are higher energy stores than Glycogen (4)
Liver could not use ketone bodies as energy source.
III- Explain briefly the following (9 marks) (1)The overall efficiency of Glucose
oxidation is about 30 % (2) Protein degradation by ubiquitination method (3) FADH2
structure and function
IV- With structural formula explain the following (12 marks)
(1)The Oxaloacetate Asp. shuttle (2) Urea cycle and its linkage to Krebs cycle (3)
Gamma-Glutamyle cycle(4) Glycerol Conversion to pyruvate (5) Acetyl CoA
carboxylation mechanism.
V- Without structural formula explain the following (Answer six) (12 marks)
(1) High proteins intake can contribute to obesity. (2) Gluconeogenesis (3) Transport of
AcetylCoA to cytosol (4) Ketogenesis (5) Conversion of propionylCoA to Glucose(6)
Regeneration method of GSH from GSSG. (7) One example about Cooperatively
between organs in metabolism
VI- Write net equation: (1) Urea Cycle. (2) Nonoxidative part of the pentose shunt. (3)
Complete oxidation of Palmitic acid (4) Palmitic acid biosynthesis, (5) Krebs cycle.
IUG , Faculty of Science,
Biological Sciences, Master program
Advanced Plant Biochemistry Bisc 6328,1st Semester,
Final Exam, 24/1/2014
Answer the Following Questions
I)Prove experimentally Four of the following:
1- Majority of chlorophyll molecules are not a part of the reaction center (exp. Of R. Emerson and
W. Arnold, 1932)
2- The probability of energy transfer in the antennae by light absorption and fluorescence.
3-Plants have two reaction centers (R. Emerson Exp., 1957).
4-Suggested # of protons/ATP formation.
5-Light reaction is coupled to CO2 and Pi assimilation.
II)Compare without equations between (Solve five)
1- Photosynthetic systems of purple bacteria and green sulfur bacteria.
2- PQ and PC
4-RPPP and OPPP and which one is preferred by the plant cell.
5-Carboxylation and oxygenation by RubisCO enzyme which one is preferred by the plant cell.
6-C3 and C4 plant metabolism.
III)Explain five of the following (without structural formula).
1-The net stiochiometrical equation of photosynthetic light reaction assuming Q cycle.
2-Noncyclic photosynthetic light reaction
3-Sources of NADH in the peroxisome used for reduction of Hpyruvate (malate oxaloacetate
shuttle).
4-Chemiosmotic hypothesis and prove it by two different experiments.
5-Mechanism of stomata opening. 6-Principle of CAM.
7-Protocol of isolation of intact chloroplast and formation of micelle crystal structure of their
membrane proteins.
IV)Explain with structural formulas (Solve four)
1-Aldol reaction mechanism. 2-Regeneration of RBP from TOP.
3-Fixation of ammonium in the chloroplast. 4-Transamination mechanism.
5-Mechanism of action of glycine decarboxylase complex
V)Ask yourself from your presentation a core question and answer it.
IUG , Faculty of Science, Dept. of Chemistry and Biochemistry,
Biochemistry A 2301, 8/4/2015, Midterm Exam
Answer the Following Questions
First (13 marks): : Complete
1-Some denaturizing agents of proteins are ------------, -----------------, and -------------.
2-Amyloidal disease such as ----------- results from accumulation of ----------------.
3- Some protein functions are-------------------, --------------------, ---------------------.
4- Acidic drugs such as Aspirin are absorbed from --------- whereas basic drugs such as
morphine are absorbed from ---------------.
5- Characteristics of peptide bond are ----------------, ---------------, and -----------------.
6- Tyrpsin cleaves a polypeptide beyond ------------------, and --------------------.
7- Amino acid analyzer determines AAs composition which includes -------------, -------8- Polypeptide fragmentation followed by Edman's reagent (PITC) determines -----------9- Beta- sheet and Alfa- helix are stabilized by -------------.
10-
Main Amino acid that disrupts Alfa-helix is ------------------------.
11-
Major bonds which stabilize 3 structure of a globular protein are ----------,----------.
12-
Amphipathic compounds have ----------------------- part, and -------------------- part.
13-
Simple lipids mainly become rancid by two methods which are ----------------, ------
14-
Major functions of Essential fatty acids are -------------, -------------------, -----------
15-
Major types of lipoproteins are: -----------, --------------, ----------, ------------.
16-
Major functions of cholesterol are ------------------, -----------------, ---------------
Second (7 marks):1-Sketch a titration curve of Ala where pK1 is 2.3, pK2 is 9.1 with
showing pI and buffer regions.
2-Draw oxygen dissociation curve of Hb with normal, high and no 2,3-BPG.
Third (7 marks): Complete the following tables: 1-Compare between Hb and Mb
Parameter
Hb
# of oxygen molecules can bind
# of polypeptide
P50
Type of oxygen dissociation
curve
Binding of CO
3- Classify as polar/ non-polar amino acid
Amino acid Polar/or non-polar
Ala
Cys
Glu
Asn
MB
Fourth (5 marks):: Complete the following reactions
1- 3 fatty acids + glycerol (Esterase) =
2- Lecithin + H2O ( lecithinase) =
3- Oil + oxygen ( long boiling) =
4- Oil + H2 ( Ni) =
5- Cys + Cys (Oxidation) =
Fifth (8 marks):: 1- Write General formulas for a dipeptide, Waxes, P.L., Sulfitides.
2- Draw the structural formulas of the following compounds
Linoleic acid, Acrolien, Phosphotidic acid, cardiolipin.
IUG , Faculty of Science
Dept. of Chemistry and Biochemistry, Midterm Exam
Proteins and Lipids biochemistry (Bioc 4308)
1st Semester, 19/4/2015
Name---------------------------------------------------------------------------------------No.-----
Answer the Following Questions
First: Without structral formula, Explain briefly
1- Importance of HbA1c determination.
2- Albumin is a poor index of a cute malnutrtion.
3- Recent steps of primary structure determination of a protein
4- Creatine function
5- Importance of oxidative deamination of Glu in the liver mitochonderia.
6- Major functions of PLP-containing enzymes
7- Urine is slightly acidic.
8- Sources of Gln released from muscles during starvation.
9- Types and function of bile acids
10- Majer functions of Met.
Second: With structral formula, write the biochemical pathways of the following
12345-
Conversion of D-aa to L- aa.
Biosynthesis of NO
Biosynthesis of Cys
Conversion Pro to Glu.
Conversion of Glucose to CO2 without passing of its intermediates in Krebs cycle.