Chapter 7: Enzyme Kinetics and inhibition

Question 1

What is enzyme kinetics?

Answer 1

Using math to describe the enzyme activity. Kcat is a catalytic rate constant. Substrate affinity: substrate and enzyme have an affinity Km.

Question 2

How can the rate of a reaction be monitored? Also known as Velocity

Answer 2

Monitored by the disappearance of the substrate or the formation of the product. There is no preferred way of monitoring the rate reaction; which every is most convenient.

Question 3

Relationship between reaction velocity and enzyme concentration

Answer 3

The slopes are important. The reaction rate and the enzyme concentration have a linear relationship.
The more enzyme present, the faster the reaction

Question 4

The parameters plotted in a hyperbolic plot

Answer 4

React with substrates in a linear fashion. The parameters plotted are the substrate concentration and reaction velocity.

Question 5

The two reactions described by the Michaelis-Menten equation

Answer 5

• Describes the hyperbolic change; velocity vs substrate plot. E= enzyme and S=substrate. E and S collide to form ES (biomolecular reaction) And ES goes back to E and S or forms P and E (both uni-molecular reactions). ES to E and P [this action is irreversible].
• The fist step is reversable and fast. The second step is slow and it is the rate determining step

Question 6

The rate equations for first and second order reactions

Answer 6

• Unimolecular or the first-order reaction; the velocity is dependent on the concentration of only one substrate.
• Look at slide 8
• Second order or the biomolecular reaction is when the velocity is dependent on two substrate concentrations. Look at slide 9

Question 7

The steady state assumption and its implications

Answer 7

• And the steady state assumption proposed that the enzyme is going to form the complex ES. Once the ES forms to some extent that amount remains virtually constant.
• b. Implications: It means that the change in the concentration of ES over time is zero. There is no change with time. [ES] has a constant value

Question 8

The rate limiting step of the Michaelis-Menten reactions

Answer 8

The rate limiting step is ES-> E +P

Question 9

The initial velocities assumption and its implications

Answer 9

• Initial velocity assumption: The ES-> E +P is not considered because it is not reversable. The K-2 can be eliminated. Look at slide 11

Question 10

The Michaelis-Menten equation and meaning of its terms

Answer 10

[E]= enzyme
[S]=Substrate

Question 11

Interpretation of the Michaelis-Menten plot

Answer 11

Each point in the plot is the velocity and the concertation of the substrate. The points will then create a best fit line.

Question 12

The definition and most common interpretation of the Michaelis constant, KM

Answer 12

• The KM is the concentration of substrate at vmax/2
• It is used to measure the enzymes** affinity for a substrate**
• The lower KM the higher the affinity.

Question 13

Meaning of turnover number or catalytic constant, kcat

Answer 13

• When there is a lot of substrate the enzyme the maximum rate can be obtained.
• Also known as the turnover number
• It indicates **how fast **an enzyme can act after it has bound its substrate.
• The rate constant of the reaction when the enzyme is saturated with substrate.
• Kcat= Vmax/[E]T because Vmax=k2[E]total

Question 14

How can the catalytic efficiency be obtained?

Answer 14

They obtained it by using the hyperbolic equation and they used the reciprocal of it.

Question 15

Lineweaver-Burk plot and how the Vmax and KM can be obtained from the plot

Answer 15

Vmax and Km: are obtained by the reciprocal initial velocity and the reciprocal concentration of the substrate. The slope will then equal the KM/Vmax.

Question 16

Types of enzymes that do not fit the Michaelis-Menten model kinetics

Answer 16

• Multiple substrates and products; 50%;They can be oxidation- reduction or transfer reactions
• Those that proceed via multiple steps; Transketolase; Its called the ping pong reaction
• Allosteric enzymes; it behaves like hemoglobin

Question 17

Characteristics of allosteric enzymes

Answer 17

• Multimeric enzymes
• Their kinetics is described by non-hyperbolic curves. Described by the sigmodal curve
• They display ** cooperative behavior **

Question 18

Meaning of cooperative behavior

Answer 18

• The binding of a substrate at one active site can affect the catalytic activity of the other sites.
• They can become more active or less active

Question 19

Interpretation of sigmoidal plot

Answer 19

• Describes the kinetics of the enzymes. The Vmax are obtained when there is a lot of substrate. But in this case the Vmax is lower and the KM has increased but the affinity is low.

Question 20

Types of enzyme inhibitors

Irresistible

Answer 20

• Inhibitor forms a covalent bond
• Some inhibitors are called suicide inhibitors
• They look different when they are bond to the enzyme

Question 21

Types of enzyme inhibitors

Reversible

Answer 21

i. Competitive inhibition; competing with the substrate to bind to the enzyme. They bind at the active site. They look like the substrate
1. The most common inhibitors
ii. Noncompetitive inhibition
1. They bind at a different site that isn’t the active site.
iii. Uncompetitive inhibition

Question 22

Why aspirin can inhibit COX

Answer 22

• Aspirin covalently modifies the COX-2 enzyme through acetylation of Ser530 near its active site, which prevents proper binding of the native substrate and thus leads to its irreversible inhibition

Question 23

5-fluorouracil can inhibit thymidylate synthase

Answer 23

5-fluorourcil is taking up byt te cells and readily converted to nucliotide 5-fluorodeoxyuridylate. It enters the active site of thymidylate synthase, where Cys-SH group adds to C6. Because of electron withdrawing F atom it prevents methylation. The inhibitor remains inthe active site, bound to cyseine side chaing rendinger thymidlate synthse inactve.

Question 24

Characteristics of competitive inhibitors and effect on Vmax and KM

Answer 24

Competitive inhibitors bind to the same site as the substrate. The KM is higher therefore there is less affinity. The Vmax will be achieved when there is a lot of substrates. The Vmax will eventually fade away and it remains the same.

Question 25

Lineweaver-Burk plot for competitive inhibition

Answer 25

It uses the same data from the hyperbolic graph. The difference is in the slope. There is a competitive inhibitor in the graph

Question 26

Characteristics of transition state analogs and effect on enzymes

Answer 26

a. Transition state analogs can be better inhibitors than substrate analogs
b. They are synthetic molecules made by chemists that are tended to mimic the proposed transitioned state.
c. The transition state binds very strongly to the enzyme

Question 27

Characteristics of noncompetitive inhibitors and effect on Vmax and KM

Answer 27

a.** Does not affect KM but decreases Vmax**
b. There is not product the Vmax is lower; no product forms because the substrate prevents the enzyme from forming the product.
c. **Metal ions act as noncompetitive inhibitors **

Question 28

Characteristics of uncompetitive inhibitors and effect on Vmax and KM

Answer 28

a. The inhibitor binds to the ES complex. They decrease Vmax and KM
b. A requirement is that it must use two substrates. One of the two substrates opens up a space in the active site of the enzyme. The inhibitor only binds after the substrate.

Question 29

The feedback inhibition mechanism

Answer 29

b. It gives back feedback by decreasing the energy. It tells it to slow down if there is too much product.
i. Ex. Phosphoenolpyruvate will give the feedback to the phosphofructokinase
ii. The accumulation in a pathway it will go back and affect the activity of one of the earlier enzymes.

Question 30

Interpretation of kinetic plots of phosphofructokinase (PFK) with and without PEP

Answer 30

With phosphoenolpyruvate, the feedback, the activity has decreased.

Question 31

phosphoenolpyruvate (PEP)

Answer 31

• Is an example of a feedback inhibitor: when its concentratino in the cell is sufficently high, it shuts down its own synthesis by blocking an earlier step in its biosynthetic pathway.

Question 32

Effect of PEP on the conformation of PFK

Question 33

The additional mechanisms for regulating enzyme activity

Answer 33

a. Rates of synthesis
b. Enzyme’s location
c. A signal may affect activity
i. Very common.
d. Covalent modification
i. Very important. See image

iv. This example is revers

Kinases catalyzed phosphorylated groups. This example is reversable