This document discusses enzyme kinetics and inhibition. It provides information on measuring the rate of enzyme-catalyzed reactions using a spectrophotometer, how different types of inhibitors (competitive, noncompetitive, uncompetitive) affect the Michaelis-Menten kinetics of enzymes, and methods for determining inhibition constants. Key topics covered include optimal conditions for alcohol dehydrogenase enzyme activity, Michaelis-Menten parameters for wild type and mutant ADH, and kinetic analysis of different ADH inhibitors like hydroxylamine, cimetidine, and formamide.
This document discusses enzyme kinetics and inhibition. It provides information on measuring the rate of enzyme-catalyzed reactions using a spectrophotometer, how different types of inhibitors (competitive, noncompetitive, uncompetitive) affect the Michaelis-Menten kinetics of enzymes, and methods for determining inhibition constants. Key topics covered include optimal conditions for alcohol dehydrogenase enzyme activity, Michaelis-Menten parameters for wild type and mutant ADH, and kinetic analysis of different ADH inhibitors like hydroxylamine, cimetidine, and formamide.
This document discusses enzyme kinetics and inhibition. It provides information on measuring the rate of enzyme-catalyzed reactions using a spectrophotometer, how different types of inhibitors (competitive, noncompetitive, uncompetitive) affect the Michaelis-Menten kinetics of enzymes, and methods for determining inhibition constants. Key topics covered include optimal conditions for alcohol dehydrogenase enzyme activity, Michaelis-Menten parameters for wild type and mutant ADH, and kinetic analysis of different ADH inhibitors like hydroxylamine, cimetidine, and formamide.
This document discusses enzyme kinetics and inhibition. It provides information on measuring the rate of enzyme-catalyzed reactions using a spectrophotometer, how different types of inhibitors (competitive, noncompetitive, uncompetitive) affect the Michaelis-Menten kinetics of enzymes, and methods for determining inhibition constants. Key topics covered include optimal conditions for alcohol dehydrogenase enzyme activity, Michaelis-Menten parameters for wild type and mutant ADH, and kinetic analysis of different ADH inhibitors like hydroxylamine, cimetidine, and formamide.
Study online at https://quizlet.com/_9ju6wu
1. What is the function of a spectrophotome- Measure the amount of light
ter? that passes throught the cu- vette
2. Which wavelength is optimal to measure 340 nm
NADH?
3. Define the absorbance value displayed on It is the logarithmic ratio be-
the spectrophotometer. tween light falling on the sam- ple and passing through it, log(l0/lt)
4. What is included in the master mix? Ethanol, NAD+, and buffer
5. Why don't we add the enzyme into the mas- The reaction will start imme- ter mix? diately, and our measurement will be inaccurate.
6. What role does ethanol play in this reac- Substrate for the enzyme tion?
7. What is the function of NAD+ in this reac- Cofactor
tion?
8. Why do we need buffer in the master mix? To stabilize reaction pH
9. What is the substrate concentration in this 0.0016 mM
master mix?
10. What aspect of a reaction do they [en- Activation energy
zymes] lower, thereby increasing the reac- tion rate dramatically?
11. What is the function of the NAD+ cofactor? It acts as an electron trans- porter or electron acceptor
12. What was just shown in the animation? The enzyme-substrate com- plex was formed
13. k2 1/3 Enzyme Kinetics Study online at https://quizlet.com/_9ju6wu When the reaction produces no intermedi- ates, which of the following rate constants will be equal to kcat?
14. Calculate the ethanol volume for two master 0 mL and 0.106 mL mixes with final substrate concentrations of 0 mM and 0.0053 mM in a final volume of 1 mL.
15. Which part of the curve should you use to A
measure the intial reaction rate?
16. What is the optimal enzyme concentration? 0.1 mg/mL
17. What is the optimal pH for the alcohol dehy- 8.5
drogenase enzyme?
18. What is the optimal temperature for the al- 44°C
cohol dehydrogenase enzyme?
19. How can we convert this into product con- A = e • c • l
centration?
20. What values do we need to calculate to plot Initial reaction rate
Michaelis-Menten curve?
21. What is the estimated Vmax for wild type 18 ¼
M/min ADH?
22. What is the estimated Vmax for mutant 480 ¼
M/min ADH?
23. Looking at the plot, which one is Km? A
24. What is the Km for wild type ADH? 16 ¼
M
25. What would the Km be for the mutant ADH? 1.9 mM
26. What would explain the difference observed A base change in the DNA in the two enzymes?
2/3 Enzyme Kinetics Study online at https://quizlet.com/_9ju6wu 27. When analyzing inhibition kinetics, which Lineweaver-Burk plot is useful in determining the type of in- hibition taking place?
28. What kind of inhibitor is Hydroxyalime? Competitive
29. In competitive inhibition, how are the Unchanged; increased
Vmax(app) and the Km(app) affected?
30. What is the Vmax(app) value for the Hydrox- 480 ¼
M/min ylamine inhibition?
31. How can you calculate the Ki for a compet- By comparing the slopes of itive inhibition? Lineweaver-Burk plots at dif- ferent inhibitor concentration
32. What is the Ki value for Hydroxylamine? 30 ¼
M
33. What kind of inhibitor is Cimetidine? Noncompetitive
34. In noncompetitive inhibition, the Vmax(app) Decreased; unchanged
is ... and the Km(app) is ...
35. What is the apparent Vmax value at 500 ¼
M 240 ¼ M/min cimetidine?
36. What is the Ki value for the Cimetidine inhi- 500 ¼
M bition?
37. What kind of inhibitor is Formamide? Uncompetitive
38. In uncompetitive inhibition, the Vmax is ... Decreased; decreased
and the Km is ...
39. What is the Km(app) value for the 100 ¼
M 1 mM Formamide inhibition?
40. What is the Ki value for the Formamide in- 100 mM
