It is all in the enzymes

Transcription

1 Enzyme regulation 1

2 It is all in the enzymes Enzymes can enhance the rates of metabolic (or other) reactions by many orders of magnitude. A rate enhancement of means that what would occur in 1 second with an enzyme s help, would otherwise require 31,710,000,000 years to take place. So essentially without enzymes such reactions don t take place. Thus, regulation of enzymatic activity is in a sense, regulation of metabolism, or any other cellular process. 2

3 Regulation and control of enzyme activity 1. Substrate level control. Most enzymes are highly tuned towards their substrates, having a very low K m. Since often [S] > K m, the change in substrate concentration does not change the reaction rate appreciably. Thus, controlling a metabolic flux is not normally achieved by varying substrate concentrations. A notable exception is glucokinase (Hexokinase IV) in the liver which has a very high K M which is roughly comparable to blood glucose concentration. This enzyme was thought to be the glucose sensor in the body. 3

4 Regulation and control of enzyme activity 2. Cooperativity, the 2 nd secret of life Cooperativy is the change in the response of an enzyme to changes in substrate concentration. It can be both negative as well as positive according to the sequential model. Binding of the 1 st substrate molecule differs from binding subsequent molecules. 4

5 Cooperativity: not only for multiple subunits (e.g. glucokinase): When [S] is low, the low affinity form of the enzyme (E ) predominates. When [S] is high, the high affinity form of the enzyme (E) predominates. The enzyme interconverts between the two states slowly. 5

6 Regulation and control of enzyme activity 3. Allosteric effectors. 6

7 Regulation and control of enzyme activity 4. Substrate cycles. The flux S 2 -> S 1 is 100 units/sec. The flux S 1 -> S 2 is 98 units/sec. The overall flux S 2 -> S 1 is 2 units/sec. If enzyme E S 2->S1 is activated by 50% (150 units/sec), then the overall flux is now 52 units/sec, an enhancement of 26 fold. Although a substrate cycle is wasteful in terms of energy expenditure it does allow moderate enzymatic activations to result in dramatic flux increases. E S 1->S2 S 1 S 2 E S 2->S1 7

8 Regulation and control of enzyme activity 5. Covalent modification. 8

9 Regulation and control of enzyme activity 6. Enzyme concentration. 9

10 Looking for Traditional Metabolic Control Points: 1. Enzymes with low Vmax. Any enzyme that is working slowly is obviously a bottle-neck in the reaction. Therefore activation of a slow enzyme can increase the flux of the entire pathway. In heart muscle glycolysis the slowest enzymes are: Hexokinase. Phosphofructokinase. Aldolase. Enolase. 10

11 2. Enzymes that catalyze reactions that are far from equilibrium. Any enzyme that is catalyzing a reaction that is essentially irreversible (i.e. far from equilibrium) can be viewed as a gate to a dam (any enzyme that isn t, cannot be used for control). This is true for: Hexokinase. Phosphofructokinase. Pyruvate kinase. Glucose transport. While glucose transport is close to equilibrium, once inside the cell, glucose is rapidly phosphorylated. 11

12 3. Cross-over points points. Upon stimulating a system, look at what happens to the concentrations of every metabolite. Any metabolite prior to the control point will be depleted and any metabolite after will be accumulated. Metabolite Concentration Difference A B C D E F G H Stimulated Unstimulated 12

13 4. Control points at the start of a pathway and immediately after brach points. Any enzyme that catalyzes the 1st step in a pathway is a potential control point since it shows commitment to the pathway. Phosphofructokinase is the obvious point in glycolysis. 13

14 Metabolic control analysis The relationship between the properties and characteristics of a metabolic pathway: Flux. Metabolite concentration. Enzyme activity. Enzyme concentration. The system studied should be in steady state. The analysis defines several variables in the system. 14

15 Enzyme flux control coefficient (C) The fractional change in the pathway flux (J) due to a fractional change in the enzyme s concentration. Flux control coefficient is a property of the pathway. The sum of all the flux control coefficients in a pathway should be equal to 1. Flux E i concentration C J Ei J J [E ] i [E ] i = J [E ] [E i] J i 15

16 Elasticity coefficient (ε) The fractional change in the enzyme s activity in response to a fractional change in the concentration of a substrate. All other components are held in place. The elasticity coefficient is a property of an individual enzyme, not a pathway. For substrates and activators ε > 0, and ε < 0 for products and inhibitors. S 1 V 1 S 2 ε S1 E 1 V i Vi S i Si = V i S i S i V i 16

17 Response coefficient (R) The response of a system to external effectors, or metabolites. The response coefficient of metabolite X on the flux J where X acts on enzyme I, is the product of the flux control coefficient and the elasticity coefficient with respect to X. A pathway flux will only respond to an effector if it acts on an enzyme with a relatively large flux control coefficient. R X J C i J ε X i 17

18 Metabolic control analysis: advantages Test the influence of an enzyme in regulating a particular pathway. Dispels simplistic notions regarding the control of all enzyme that catalyze reaction which are far from equilibrium. Important application in biotechnology, where one wants to increase a particular pathway. Helps in pointing out enzymes with high flux control coefficient as potential drug targets. 18

19 MCA example worked out: 1. Flux control coefficient Here are the results from an experiment in which the rate of ethanol production by brewers yeast was tested as a function of expression levels of the last enzyme of the pathway: alcohol dehydrogenase. 250 [Alcohol dehydrogenase] Ethanol production Ethanol production Concentration of alcohol dehydrogenase

20 J C Ei J J [E i ] [E i ] ΔJ J Δ[E i ] [E i ] [Alcohol dehydrogenase] Ethanol production J Point 1 C Ei = = 0.4 J Point 3 C Ei = = 0.14 J Point 2 C Ei = = 0.5 J Point 4 C Ei = = 0 20

21 MCA example worked out: 2. Elasticity coefficient When isolated alcohol dehydrogenase is incubated in ethanol concentrations which are 20% above normal, it is inhibited by 10%. V i ε E 1 Vi S1 S i Si ΔV i Vi ΔS i Si = 10% 20% =

22 MCA example worked out: 3. Response coefficient When isolated alcohol dehydrogenase is incubated in ethanol concentrations which are 20% above normal it is inhibited by 10%. R X J C i J ε X i = =

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24 Aspartate transcarbamoylase 24

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32 Another subunit 32

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38 ATP: purine vs pyrimidines More ATP ore RNA synthesis 38

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