Enzyme Kinetics

Question 1

What are three ways that enzymes stabilize transition?

Answer 1

Positioning of acid-base groups to transfer protons to or from transition state intermediate

Having charged groups or metal ions to stabilize developing charges

Imposing steric strain on substrates so that their geometry approaches that of a transition state.

Question 2

How do enzymes effect the kinetics of a reaction?

Answer 2

Reduce the activation energy required for the reaction.

No effect on Keq (equilibrium)

Question 3

How do enzymes lower the free energy of activation for a reaction?

Answer 3

By binding the transition state of the reaction better than the substrate.

Question 4

What is a zero order reaction?

Answer 4

Rate of reaction is independent of substrate concentration

Proportional to enzyme concentration of some other factor (e.g. temp, pH)

Question 5

What is a first order reaction?

Answer 5

The reaction rate is proportional to the concentration of the substrate

Question 6

What is a second order reaction?

Answer 6

The rate of the reaction is proportional to the concentration of 2 substrates.

Question 7

What is a Pseudo-first order reaction?

Answer 7

The rate appears to depend on concentration of only on substrate, but two are involved in the reaction

Occurs when one substrate is in very high concentration, but the other is low

E.g. when water is a substrate

Question 8

What is assumed in Michaelis-Menten enzyme kinetics?

Answer 8

ES complex is in rapid equilibrium with free enzyme E

Breakdown of ES to form products is slower than the formation of ES and breakdown of ES to E+S

Question 9

What is Km?

Answer 9

The substrate concentration that corresponds to 1/2Vmax.

Question 10

Describe the effect of substrate concentrations on Enzyme-Catalyzed Reactions

Answer 10

At low [S], the reaction is first order, as [S] increases the velocity slopes from first order to zero order at high [S].

Question 11

What are Lineweaver-Burk plots/equation used for?

Answer 11

Analysis of two-substrate data or inhibition.

Good technique to calculate Vmax and Km values accurately

Question 12

What is the slope of a LB plot?

Answer 12

Km/Vmax

Question 13

What is the y-intercept of a LB plot?

Answer 13

1/Vmax

Question 14

What is the x-intercept of a LB plot?

Answer 14

-1/Km

Question 15

What does the magnitude of the Km indicate?

Answer 15

Small Km - tight binding

High Km - weak binding

Question 16

What is the turnover number (Kcat)for an enzyme?

Answer 16

The number of substrate molecules converted to product per enzyme molecule per unit of time.

High Kcat means the enzyme is very efficient

Question 17

What is Kcat/Km and what does it indicate?

Answer 17

The specificity constant.

Indicates how good an enzyme is for a given substrate.

10^8 are perfect.

Question 18

Describe the effect of pH on enzyme activity.

Answer 18

Most have a pH optimum - usually reflects physiological conditions.

Extreme pH levels will cause denaturation.

Question 19

What is the effect of temperature on an enzyme?

Answer 19

Activity increases with temperature—usually doubles with 10C rise.

Full denaturation usually occurs at 70C

Question 20

Describe the two types of sequential, or single-displacement reactions for enzymes.

Answer 20

• Both substrates must bind in a specific order to form ternary complex
• Both substrates can bind in any order to form ternary complex.

Reaction will only proceed when both substrates are bound.

Question 21

Describe the Ping-Pong, or double-displacement mechanics for multi-substrate enzymes.

Answer 21

First substrate binds and reaction completes, leaving behind a different form of the enzyme that can catalyze a second reaction with a different substrate, restoring the original version of the enzyme.

No complex formation

Question 22

What do intersecting lines in a LB plot of bisubstrate reactions indicate?

Answer 22

The formation of a ternary complex, and therefore, a single-displacement reaction.

Question 23

What do parallel lines on a LB plot of bisubstrate reactions indicate?

Answer 23

A Ping-Pong (double-displacement) mechanism

Question 24

What are enzyme inhibitors?

Answer 24

Chemicals that reduce the rate of enzymatic reactions.

Question 25

What are the two major types of inhibitors?

Answer 25

Reversible and Irreversible

Question 26

What are the two types of reversible inhibitors?

Answer 26

Competitive and noncompetitive.

Question 27

How does competitive inhibition affect enzyme kinetics?

Answer 27

Decreases Km

No change in Vmax

Can be overcome by increasing [S]

Question 28

What is the effect of noncompetitive inhibition on enzyme kinetics?

Answer 28

Decreases Vmax

Does not affect Km

Question 29

What causes mixed inhibition?

Answer 29

The dissociation constant for S from ESI differs from that of ES.

Ki is not equal to Ki’

Question 30

How does mixed inhibition affect enzyme kinetics?

Answer 30

Changes both Vmax and Km

Question 31

What is methotrexate and how does it function?

Answer 31

Strong competitive inhibitor of dihydrofolate reductase.

Shuts down DNA synthesis, used in cancer treatment

Question 32

How does arsenite function?

Answer 32

Noncompetitive inhibitor of lipoamide-containing enzymes.

Question 33

What are statins and how do they work?

Answer 33

Cholesterol lowering drug class

Competitive inhibitors of HMG-CoA reductase

Question 34

How do B-Lactam antibiotics function?

Answer 34

Irreversible inhibition of Transpeptidase in bacteria.

Question 35

How does Fluorouracil work?

Answer 35

Irreversible inhibition of Thymidylate Synthase.

Used to treat actinic or solar keratoses and skin cancer