Lecture 14: Enzyme-substrate interactions

Question 1

Models of enzyme behaviour‘Michaelis-Menten’ kinetics

Answer 1

Simple models assume the following:

Only one molecule of one substrate binds to the enzyme (One substrate reactions are rare – isomerizations and rearrangements);

Enzyme and substrate form a [ES] complex (rate-limiting step);

Enzyme converts substrate to product and product release occurs (fast);
Products bind weakly to enzymes.

Question 1

Factors that affect enzyme activity

Answer 1

pH – due to changing ionisation state of side-chains. Grossly inappropriate pH unfolds proteins;

Denaturing reagents (detergents, urea, guanidinium hydrochloride);

Temperature – higher temperatures increase rate until thermal denaturation (unfolding) of the protein occurs;

Activity is proportional to amount of enzyme;

Substrate concentration (Lecture 14; this lecture);

The presence of inhibitors

Question 2

[E]

Answer 2

concentration of enzyme

Question 3

[S]

Answer 3

concentration of substrate

Question 4

[ES]

Answer 4

concentration of enzyme-substrate complex

Question 5

[P]

Answer 5

concentration of product

Question 6

v

Answer 6

rate

Question 7

Vmax

Answer 7

rate when all enzyme active sites are occupied (the enzyme is saturated

Question 8

Km

Answer 8

[S] at which v = ½ Vmax

Question 9

At high [S],

Answer 9

ate (v) does not increase with further increases in [S] (zero order). Rate only depends on [ES] and k3 (i.e., saturation has occurred

Question 10

Assumptions of steady-state model

Answer 10

[E] &laquo_space;[S] (typically nM vs. mM);
[S] remains constant; Measured rate of reaction is linear at beginning of curve (‘initial rate’);
[ES] remains the same throughout course of reaction (‘The steady-state approximation’);
Product binds weakly to enzyme;
Rate of conversion of product to substrate is negligible

Question 11

The Lineweaver-Burk Plot

Answer 11

Plot 1/v against 1/[S]

Y-intercept = 1/Vmax
X-intercept = -1/Km

Higher precision;

Lower accuracy;

Errors are not equal at all points
(least squares regression is not appropriate

Question 12

Meaning of kinetic parameters

Answer 12

Km = concentration of substrate at which v = 0.5 Vmax.

Km is NOT a substrate binding constant, but is often interpreted as a crude measure of affinity of enzyme for substrate;

Km often similar to substrate concentration in cell;

Vmax = maximum velocity = limiting rate (e.g., in nmoles product/min);

kcat = Vmax/amount of enzyme in e.g., nmoles. This is the first order rate constant for conversion of substrate to product. In our model kcat = k3

Question 13

Mode of action of inhibitors

Answer 13

Inhibitors can resemble the substrate in structure;

Inhibitors affect either Km, Vmax or both;

Potency is measured by Ki value (concentration of inhibitor that causes a 2-fold change in Km or Vmax);

Ki values related to binding energy (ΔG);

Smaller Ki numbers mean tighter binding (higher potency);

Question 14

Kcat

Answer 14

Vmax/moles of enzymes

Question 15

Competitive inhibition

Answer 15

The most common form of inhibition;

Km increases (X-intercept changes);

Km (I) = Km (1 + [I]/Ki)

No change in Vmax (Y-intercept the same);

Inhibition decreases with increased substrate concentration.

Question 16

Non-competitive inhibition

Answer 16

Uncommon for single substrate enzymes;

Vmax is reduced;

Vmax/Km (I) = Vmax/Km(1 + [I]/Ki);

Km is not affected;

Inhibition not reduced by increasing substrate.

Question 17

Mixed competitive inhibitor

Answer 17

Common form of inhibition;

Km is increased (as for competitive inhibition);

Vmax is decreased (as for non-competitive inhibition);

α factor defines the competitive and non-competitive components.

Question 18

Uncompetitive inhibition

Answer 18

Uncommon mode of inhibition;

Inhibitor binds to the ES complex;

Parallel lines in Lineweaver-Burk plot;

Both Km and Vmax reduced by same amount, (1 + [I]/Ki);

Increasing substrate concentration increases inhibition.

Question 19

IC50

Answer 19

concentration of drug required for 50% reduction in activity

Question 20

Tight-binding Inhibitors

Answer 20

Tight-binding inhibition occurs when the concentrations of (active) enzyme and inhibitor are similar;
This means that the concentration of free inhibitor does not approximate to total inhibitor (unlike in the cases above);
The apparent Ki value will increase with increasing enzyme concentration;
Tight-binding inhibition often has very slow onset, and the enzyme inhibitor complex can have a very long half-life (Ki = koff/kon);
This means that infrequent dosing regimens can be used (easier for patient, reduced side-effects etc.). Therefore, many drug discovery programmes aim to develop a tight-binding inhibitor.