Kinetics of Enzyme Reactions
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Rate and order of chemical reactions, kinetics of zero order and 1st order reactions. Mechanism of enzyme catalyzed reactions, activity and catalytic concentration of enzymes, factors affecting the enzyme activity, Michaelis plot, Km, inhibitors.
Quantification of Enzymes
Expression of enzyme quantity	Unit	Dimension
Catalytic (enzyme) activity	Katal (kat)	
	International unit (U, IU)	umol/min
Catalytic concentration		
Mass concentration	g/L	
1.   Derive the relation between the values of catalytic activity in ukat and 1U and vice versa.
Determination of Catalytic Activity
[P]
10
15 t (min)
E
3» P
catalytic concentration =
A[P] A/
2.  Characterize the main ways of catalytic activity measurement a) Kinetic method .........................
b) Constant time method
c) Immunochemical determination ............................................................
v
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3. Lactate dehydrogenase has the catalytic activity 2 (.ikat. How many molecules of lactate will formed from pyruvate in one minute, if an excess of substrate is present? (7.23-io1
4. How much product will be formed in an enzymatic reaction in 10 minutes, if the enzyme activi is 10 ukat? What experimental conditions must be kept to achieve the amount theoretica calculated? (6 mmo
5. Serum (0.1 mL) was added into a reaction mixture. After 10 minutes, exactly 6• 10"3 mmol of t product were determined. What is the catalytic concentration of the enzyme? Is the result differen from the activity determined by the kinetic method? (ioo ukat/L,
6. The reaction mixture contains 2.5 mL of a buffer, 0.2 mL of NAD+ solution, 0.1 mL of serum an 0.2 mL of lactate solution. The reaction proceeded exactly for 10 minutes and 0.0012 mol/L o NADH was measured in the mixture. Calculate the catalytic activity and the catalyti concentration of lactate dehydrogenase. (6 nkat, 60 ukat/L
7. Calculate the activity of catalase, if 6.72 uL Ot was released in 10 minutes. In the reaction mixtur was an excess of H202 and the reaction proceeded under normal conditions. (i nkat
Optical (UV) Test
NAD(P)+    NAD(P)H + H+ S   ^T..:,.^...^... P NAD(P)H + H+ NAD(P)+
, v0
260
340
\ (nin)
Catalytic concentrat ion
Use:
a) Determination of enzyme activities -NAD(P)-dependent dehydrogenases
- coupled determination of other enzyme activities
b) Determination of substrate concentration - e.g. lactate
8.  How will the absorbance at 340 nm change during the lactate determination when using the optical test?
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Factors Affecting Rate of Enzyme Reactions
I. Substrate Concentration
The course of a mono-substrate enzyme reaction:
E + S   « ES -►   E + P
Michaelis Plot (Saturation Curve)
For steady state: |ES] = constant
9. Compare:  kinetic curve: dependence of...............on..................
saturation curve:       dependence of............on..................
10. Suggest an experiment, which provides the data for the construction of the saturation curve.
11. What is the dimension of A"M?
12. Characterize the three parts of the Michaelis-Menten plot for the values: a) [S] « KM
b) [S] » Km. What are the reaction orders under these conditions?
13. Characterize the point on the saturation curve for the value [S] = KM.
14. Calculate the [S]/ATm ratio, if the initial reaction rate v0 is: a) 90 % Vmax\ b) 99 % Fmax.      (a) 9; b) 99)
1 5. For which of the following substrates Si, Si a S3 does an enzyme with a broad substrate specificity have the highest affinity (KM\ = 400 umol/L, ATM2=1000 umol/L, Km = 60 umol/L)?
16. The enzyme P-galactosidase has /^(lactose) = 400 umol/L. What concentration of lactose has to be used to maintain a zero-order kinetic? (>ioo Km, i.e. >40 mmoi/L)
17. The KM of an enzyme was increased twice in comparison with the normal value due to a mutation. What concentration of substrate must be used not to change the reaction rate?
18. L-Asparagin is necessary for proteosynthesis in some cancer cells. The enzyme asparaginase converting Asn to Asp and ammonia is administered during the treatment of some leukaemia types. The decrease of Asn in circulation occurs consequently and the proliferation of cells is lowered. Which of the asparaginase forms will be the most suitable for the treatment if the Asn concentration in blood is 0.2 mM?
a) KM = 0.2 mM; Fmax = 0.1 raM/h b) KM = 0.2 mM; Vmax = 0.5 mM/h
c) A'm = 2 mM; Vmax = 0.1 mM/h d) Km = 0.1 mM; Vm% = 0.5 mM/h
19. Explain the term substrate specificity using hexokinase and glucokinase as examples.
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Saturation Curve of Allosteric Enzymes
No effector
Allosteric effector
- low molecular compound (often intermediate or product)
- binding into another region different from the active site
- change of enzyme conformation —> change of activity
Allosteric enzyme
- usually more subunits (often regulatory and catalytic)
- regulatory functions in metabolism
[si
II. Enzyme Concentration
In general:        v0 = k ■ [E]t
For a fully saturated enzyme ([E],= [ES]): v0= Vmax = kcat • [E],
where [E],= [E] + [ES];
/feat = Piwh / [E]t molecular activity (turnover number)
20. Construct the saturation curves for the three different enzyme concentrations ([E|] < [E2] < [E3]) of the same enzyme and the same reaction type. How will Km, Vmm values change?
21. The molecular activity of carbonic anhydrase is 6-10s s"\ How many molecules of carbonic acid were formed, if the reaction was catalyzed by one molecule of the enzyme for 10 milliseconds? The enzyme is fully saturated. (6 ooo)
22. The molecular activity of lysozyme is 0.5 s" . How many glycosidic bonds would be hydrolyzed by 1 umol of the enzyme in 1 minute? Assume that the enzyme would be saturated. (o.5Mmoi=3.oiio"j
III. Temperature
IV. pH
V. Activators
23. In which way is the enzyme activity affected by: a) temperature; b) pH; c) activators? Give examples.
24. What is Taq polymerase, at which method it is used?
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VI. Inhibitors
25. Explain the difference between the reversible and irreversible inhibition.
26. Give examples of irreversible inhibition.
27. What types of irreversible inhibition are distinguished?
28. Complete into the table, how are changed the values of KM and Vmax and give examples of inhibitors.
Parameter	Competitive inhibition	Non-competitive inhibition
		
V ' max		
Inhibitor		
29. Draw the course of competitive and non-competitive inhibition. Mark the values KM, Kmax into the plots.
a) competitive inhibition b) non-competitive inhibition
i = 0
30. Explain the terms: proenzyme, isoenzyme and isoform.
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