Section 8

Enzyme Kinetics

1. Produce a reading log for the sections in your text that discuss the Michaelis-Menten equation, including
kcat.
2. Focus on the derivation of the Michaelis-Menten equation. List and explain the assumptions underlying the
Michalis-Menten equation. Provide definitions for each term.
3. What is equal at equilibrium?
4. What is the general expression Keq (the equilibrium constant) in terms of product and reactant concentration?

Why
Enzymes catalyze most reactions that occur in biological systems and thus control the rates of these reactions.
Examining the kinetics of an enzyme catalyzed reaction helps us learn about how enzymes function. In this
activity, you will learn about the kinetic parameters that biochemists use to characterize enzymes and the
graphical methods that allow biochemists to obtain kinetic parameters from experimental data.

Outcomes
1. Be able to articulate and apply what the enzyme parameters of KM, Vmax, kcat and kcat /KM tell us about the
enzyme.
2. Know the Michaelis-Menten rate equation and be able to utilize the rearranged versions of the MichaelisMenten equation to determine the values for KM and Vmax.
3. Use KM and Vmax values to discuss qualities of enzymes related to their catalytic properties.

Plan
1. Form enzyme teams. Assign roles of manager, reflector, recorder, spokesperson.
2. Answer the Critical Thinking Questions.
3. Share two insights your team made about enzyme kinetics today.

Critical Thinking Questions

1. Recall your study of equilibria and kinetics from general chemistry. You used equations with upper case K
and lower case k during the study of equilibria and kinetics respectively. What do the upper and lower case
letters refer to?

39

Equation 1

Km
1
1
=
+
Vo Vmax [ S ] Vmax

Equation 2

E+S

k1
k 1

E S 2 E + P

Velocity, mM product/min

Graph 1

4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0

10

15

[S]. mM

2. Compare your answers from your assignment and take only two minutes. Discuss the assumptions made
during the derivation of the Michaelis-Menten equation. Be sure to consider assumptions pertaining to
[ES], rate of product formation, [S], and number of substrates per reaction.

3. Define the term Vmax in your own words

4. Solve the Michaelis-Menten equation for KM when V0 = Vmax /2. What does this tell you about the relationship
between [substrate] and enzymes with high KM values? With low KM values? Make a generalization.

5. a. What is the KM value as you can estimate it from Graph 1?

b. On Graph 1, draw a curve that has the same Vmax but a larger KM value.
6. Examine Equation 2. When k2 is very small compared to k-1 the KM becomes an approximation of the affinity
of enzymes for substrate. Explain why this approximation makes sense. Does a large KM indicate high or
low affinity of enzyme for substrate?

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Foundations of Biochemistry

7. When k2 is very small compared to k1 and k1, what process is rate determining in product formation?

8. Many enzyme-catalyzed processes are multi-step reactions.

a. In a multi-step reaction, what step determines the overall reaction rate?

b. What step in M-M kinetics determines overall reaction rate?

c. The term used for the overall rate constant for multi-stp reactions is kcat. Discuss why it makes sense
that kcat = k2 in simple Michaelis-Menten reactions.

9. In simple M-M kinetics, the units of kcat are s1. Discuss how the units are consistent with the name turnover
number. If kcat is large, what does that imply about the enzyme?

10. The term efficiency is often used in describing enzymes. What does it mean for an enzyme to be efficient?

continued on next page

Section 8 Enzyme Kinetics

41

11. How is your answer to question 9 consistent with the term for catalytic efficiency (kcat /Km)? Discuss the
contribution of the terms kcat and KM to the overall term of catalytic efficiency.

12. Discuss the meaning of enzyme reaction mechanism in the context of catalysis. How does the study of
enzyme kinetics relate to reaction mechanism?

Consider the enzymes in the table below and answer the following questions. Answers should be brief (1-3
sentences).
Enzyme

KM(M)

kcat(s-1)

9.5 10-5

1.4 104

2.5 10-2

1.0 107

5.0 10-6

8.0 102

1. Which enzyme has the highest affinity for substrate? How do you know?

2. Which enzyme converts the most substrate to product in a given period of time? How do you know?

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Foundations of Biochemistry

3. Which enzyme has the highest catalytic efficiency? How do you know?

catalytic
triad

Ser 195
His 57

N
|

H
O

Gly 193 NH
R

N
|

C
C

NH

O
R

A diagram of the enzyme active site for

chymotrypsin is shown. The amino acids
Gly 193, Ser 195, His 57 and Asp 102
form part of the active site. The amino
acids Ser 195, His 57 and Asp 102
comprise what is known as the catalytic
triad. A portion of a natural peptide
substrate from R to R is depicted with
the residue phenylalanine occupying the
hydrophobic pocket of the enzyme.

Asp 102

4. Chymotrypsin is a serine protease enzyme. The KM for the reaction of chymotrypsin with N-acetylvaline
ethyl ester is 8.8 10-2 M, and the KM for the reaction of chymotrypsin with N-acetyltyrosine ethyl ester is
6.6 10-4 M.
a. Which substrate has a higher apparent affinity for the enzyme?

b. Propose a reason for the difference in affinity based on the shape of each of the substrates (see active
site figure, chymotrypsin cleaves on the C-side of aromatic residues).

c. Which of the substrates is likely to have a higher Vmax?

Section 8 Enzyme Kinetics

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Foundations of Biochemistry