Ch. 7 enzyme kinetics and inhibition

Question 1

What is enzyme kinetics?

Answer 1

• It’s the use of math to fully describe enzyme activity by quantifying an enzyme’s catalytic power, substrate affinity and its response to inhibitors

Question 2

How can the rate of a reaction be monitored?

Answer 2

-The rate of a reaction which is also known as velocity can be expressed as the disappearance of substrate or the formation of product. (i.e when glyceraldehyde-3-phosphate reacting with triose phosphate isomerase

Question 3

Relationship between reaction velocity and enzyme concentration?

Answer 3

-The reaction velocity and enzyme concentration have a linear relationship. When concentration doubles the rate doubles. As well as the more the enzyme is present the faster the reaction

Question 4

The parameters plotted in a hyperbolic plot

Answer 4

-The parameters plotted in the hyperbolic plot are reaction velocity (y-axis) and substrate concentration (S). When you double the concentration of the substrate, the reaction doesn’t increase.

Question 5

The two reactions described by the Michaelis-Menten equation

Answer 5

-Bimolecular: where E and S collide to form ES
- Unimolecular: ES goes back to E and S or forms P and E

Question 6

The rate equations for first and second order reactions

Answer 6

-Rate equation for a 1st order reaction is v=k[A]
-Rate equation for a 2nd order reaction is v=k[A][B]

Question 7

The steady state assumption and its implications

Answer 7

-it is used to derive Michaelis-Menten equation, The ES complex forms from the enzyme and has a constant value. The reversible reaction of the 2nd step is not included. Some of the reaction decomposes and some reacts. Change of concentration ES/ Change in time=0

Question 8

The rate-limiting step of the Michaelis-Menten reactions

Answer 8

The rate-limiting step of the Michaelis-Menten reaction is ES—> E+P

Question 9

The Michaelis-Menten equation and meaning of its terms

Answer 9

• V= Vmax[S]/Km + [S]
• It is a hyperbolic equation.

Question 10

The initial velocities assumption and its implications

Answer 10

-Initial velocities assumption states that K2 can be eliminated.
-V=d[P]/dt=k2[ES]

Question 11

Interpretation of the Michaelis-Menten plot

Answer 11

-Be able to describe the assumptions and initial velocity of the plot no need to derive the equation.

Question 12

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

Answer 12

-Km is derived from the m-m equation and ES is replaced with Km
-Km will be the concentration of substrate at which the substrate is half velocity at max when looking at the graph. (Vmax/2)
-Km is often used as a measure of an enzymes affinity for a substrate
-The lower the Km , the higher the affinity

Question 13

Meaning of turnover number or catalytic constant, kcat

Answer 13

-Is related to how fast the product has formed when the enzyme has bound to the substrate.
-It is the rate constant of the reaction when the enzyme is saturated with substrate.

Question 14

How can catalytic efficiency be obtained?

Answer 14

-Kcat = vmax/[E]t, since Vmax=k2[E] total

Question 15

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

Answer 15

-The Lineweaver-Burk plot linearized the M-M kinetics data. To do this they take the M_M equation and take the reciprocal of both sides
-The Lineweaver-Burk plot is linear
-Vmax is -1/km and Km is the intercept which is 1/vmax

Question 16

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

Answer 16

-Allosteric enzymes like hemoglobin

Question 17

Characteristics of allosteric enzymes

Answer 17

-Multimeric enzymes
-Their kinetics are described by sigmoidal curves
-They display cooperative behavior

Question 18

Meaning of cooperative behavior

Answer 18

-is the binding of a substrate at one active site creating a conformation change that can affect the catalytic activity of the other active sites

Question 19

Interpretation of sigmoidal plot

Answer 19

-The sigmoidal plot shows the effect of cooperative substrate binding, Vmax will be obtained no matter what (i.e if the concentration is high then Vmax will be high.
-Km shifts to the right in sigmoidal plots and increases dramatically and the affinity is low. ( for allosteric proteins the affinity is also low)

Question 20

Types of enzyme inhibitors

Answer 20

• Irreversible: Form a covalent bond and there is no way for it to go back, some inhibitors are called suicide inhibitors (drugs like aspirin and blood pressure) in which they enter the active site and begin to react like normal but they are unable to undergo the complete reaction which the substrate becomes stuck in the active site.
  -Reversible (most common): competitive inhibition meaning they compete with an enzyme or substrate, If it is a substrate it will bind to the active site, noncompetitive inhibitors bind at the allosteric site

Question 21

Why aspirin can inhibit COX

Answer 21

• Aspirin can inhibit cox because it does not catalyze the enzyme acetylate

Question 22

5-fluorouracil can inhibit thymidylate synthase

Answer 22

• When 5-fluorouracil is taken up by the cells, the deoxyribose-5-phosphate(dUMP) enters the active site of thymidylate synthase, where the Cys-SH group adds a carbon at C6. In normal conditions, this enhances the nucleophilicity of C5 so that it can accept the methyl group. But the presence of the electron-withdrawing F atom prevents this and the inhibitor remains on the active site and is bound to the cysteine side chain which then renders the thymidylate inactive.

Question 23

Characteristics of competitive inhibitors and effect on Vmax and KM

Answer 23

• Competitive inhibitors bind to the site as the substrate. The main characteristic is that coinhibitors look like the substrate
• Competitive inhibitors increase Km but do not affect Vmax
  Lineweaver-Burk plot for competitive inhibition
  -With inhibitor slope = alpha Km/ Vmax

Question 24

Characteristics of transition state analogs and effect on enzymes

Answer 24

-Are also known competitive inhibitors and can be better inhibitors than substrate analogs.
-They inhibit enzymes, in order to catalyze a reaction, the enzyme bust bind to and stabilizes or lowers the energy of the reaction’s transition state. The compound mimics the transition state to take advantage of the features in the active site.

Question 25

Characteristics of noncompetitive inhibitors and effect on Vmax and KM-

Answer 25

-Noncompetitive inhibitors do not affect Km but decrease Vmax. This is because the inhibitor doesn’t bind to the recommended site but to the inhibitor site.
- Metal ions act as noncompetitive inhibitors. (Al3+)

Question 26

Characteristics of uncompetitive inhibitors and effect on Vmax and Km

Answer 26

• In uncompetitive inhibitors, the inhibitor binds to the ES complex. When Vmax is decreased, Km is decreased.

Question 27

The feedback inhibition mechanism

Answer 27

-When concentration in the cell is sufficiently high, it shuts down its own synthesis by blocking the phosphofructokinase in an earlier step allowing phosphoenolpyruvate to bind.

Question 28

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

Answer 28

-Without PEP, PFK binds to the substrate and forms a hyperbolic plot
-With PEP, PFK forms a sigmoidal plot

Question 29

Effect of PEP on the conformation of PFK

Answer 29

-When PEP binds to the enzyme it causes a conformational change in which Arg 16 changes places with Glu 161

Question 30

The additional mechanisms for regulating enzyme activity

Answer 30

1. Rate of synthesis in which an enzyme can be regulated
2. Enzyme’s location
3. A signal may affect the activity and this is common becomes activated when there is a signal (i.e calcium which is a signal for most enzymes)
4. Covalent modification: Which phosphorylation/ dephosphorylation of glycogen phosphorylase b(less active) and glycogen phosphorylase a (more active)