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Enzyme regulation

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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 1017 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.

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Regulation and control of enzyme activity���1. Substrate level control.

•  Most enzymes are highly tuned towards their substrates, having a very low Km.

•  Since often [S] > Km, 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 KM which is roughly comparable to blood glucose concentration.

•  This enzyme was thought to be the glucose sensor in the body.

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Regulation and control of enzyme activity���2. Cooperativity, the “2nd 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 1st substrate molecule differs from binding subsequent molecules.

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Cooperativity: not only for multiple sub-units (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.

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

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Regulation and control of enzyme activity���4. Substrate cycles.

•  The flux S2 -> S1 is 100 units/sec. •  The flux S1 -> S2 is 98 units/sec. •  The overall flux S2 -> S1 is 2 units/sec. •  If enzyme ES2->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.

ES2->S1

S2

S1 ES1->S2

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

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

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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.

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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.

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A B C D E F G H Unstimulated

Met

abol

ite���

Conc

entra

tion

Diff

eren

ce

Stimulated

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.

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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.

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•  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.

Metabolic control analysis

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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.

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CEiJ ≡

∂JJ

∂[Ei][Ei]

=∂J∂[Ei]

⋅[Ei]J

Flux

Ei concentration

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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.

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→ S1→V1S2 →

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εS1E1 ≡

∂ViVi

∂SiSi

=∂Vi

∂Si⋅SiVi

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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.

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RXJ ≡ Ci

JεXi

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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.

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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.

0

50

100

150

200

250

0 50 100 150 200 250

Concentration of alcohol dehydrogenase

Eth

an

ol p

rod

ucti

on[Alcohol

dehydrogenase]Ethanol

production12 10018 12024 140100 200200 200

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[Alcohol dehydrogenase]

Ethanol production

12 10018 12024 140100 200200 200

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CEiJ ≡

∂JJ

∂[Ei][Ei]

≅ΔJ

JΔ[Ei]

[Ei]

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Point 1⇒ CEiJ =

120 −100100

18 −1212

= 0.4

Point 2⇒ CEiJ =

140 −120120

24 −1818

= 0.5

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Point 3⇒ CEiJ =

200 −140140

100 − 2424

= 0.14

Point 4 ⇒ CEiJ =

200 − 200200

200 −100100

= 0

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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%.

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εS1E1 ≡

∂ViVi

∂SiSi

≅

ΔViVi

ΔSiSi

=−10%20%

= −0.5

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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%.

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RXJ ≡ Ci

JεXi = −0.5 ⋅ 0.45 = −0.2250

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Aspartate transcarbamoylase

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Another subunit

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ATP: purine vs pyrimidines More ATP ore RNA synthesis

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