kinetic enzymes

7/28/2019 Kinetic Enzymes

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Enzyme KineticBMB Department

School of MedTarumanagara Univ


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Enzyme Kinetics

Enzymes endow cells with the remarkable capacity to

exert kinetic control over thermodynamic potentiality

Enzymes are the agents of metabolic function

What we want to be able to determine:

Maximum velocity

Substrate affinity

Inhibitor affinity

What it can tell us:

Flow through metabolic pathways

Utilization of substrates

What can we do with the information:

Control and manipulate metabolic events

Kinetics is the study of the rates of reactions


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The Michaelis-Menten Equation


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Important Conclusions of Michaels -

Menten Kinetics

when [S]= KM, the equation reduces to

when [S] >> KM, the equation reduces to

when [S]


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Important Conclusions of Michaels -

Menten Kinetics


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LineweaverBurk Double Reciprocal Plots

It is difficult to determine Vmax experimentally The equation for a hyperbola can be transformed into

the equation for a straight line by taking thereciprocal of each side

The formula for a straight line is y = mx + b

A plot of 1/V versus 1/[S] will give a straight linewith slope of KM/Vmax and y intercept of 1/Vmax

Such a plot is known as a Lineweaver-Burk double

reciprocal plot


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LineweaverBurk Double Reciprocal Plots


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Significance of Km

Km is a constant

Small Km means tight binding; high Km means weak

binding

Useful to compare Km for different substrates for one

enzyme

Hexokinase : D-fructose1.5 mM

D-glucose0.15 mM

Useful to compare Km for a common substrate usedby several enzymes

Hexokinase: D-glucose0.15 mM

Glucokinase: D-glucose20 mM


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Kinetic vs Chemical Mechanism

An enzyme kinetic mechanism is the order of

substrate addition and product release in an

enzyme catalyzed reaction

A chemical mechanism is the chemical

pathway of conversion of S P, including the

structures of any intermediates


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Bi-substrate Reactions

The MichaelisMenten model of enzyme kinetics

was derived for single substrate reactions

The majority of enzymatic reactions have multiple

substrates and products Bi-substrate reactions account for ~ 60% of the

known enzymatic reactions.


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Substrate Addition / Product Release

The order of substrate addition and product release inmost enzymatic reactions follow two reactionmechanism

Sequential reaction - all substrates must bind to the

enzyme before the reaction occurs and products arereleased

Ordered sequential

Random sequential

Ping-pong reaction - one or more products arereleased before all substrates have been added and analternate stable enzyme form, F, is produced in thehalf reaction


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1) Sequential Reaction

Ordered sequential

Random sequential


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2) Ping-pong Reaction


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Initial Velocity Plots

sequential reaction exhibits an

intersecting pattern of linesOrder and random substrate

additions cannot be distinguished

in this type of plot

Ping-pong reactionshows

parallel or non-

intersecting lines


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Influence of enzyme concentration

v = k3 [E], as

[S]>>[E]


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Influence of temperature

Optimum temperaturemost of them are in the

range from 35 to 45

.


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Influence of pH

Optimum pH


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Enzyme Inhibition

Enzyme inhibitors are important for a variety of reasons

1) they can be used to gain information about the shape on

the enzyme active site and the amino acid residues in the

active site.

2) they can be used to gain information about the chemical

mechanism.

3) they can be used to gain information about the regulation

or control of a metabolic pathway.

4) they can be very important in drug design.


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Enzyme Inhibition

Reversible inhibitor: a substance that binds to anenzyme to inhibit it, but can be released

usually involves formation of non-covalent bonds

Generally two types

Dead end

Product

Irreversible inhibitor: a substance that causes

inhibition that cannot be reversed

usually involves formation or breaking of covalent

bonds to or on the enzyme


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Inhibitors

Irreversible inhibition

Reversible inhibition

competitive inhibition

non-competitive inhibition

uncompetitive inhibition


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Irreversible inhibition

Irreversible inhibition:The inhibitor combine with essential group ofenzyme active center by covalent bond,

resulting in enzymatic activity loss.


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Inhibition Patterns

An inhibitor may bind at the same site as one of the

substrates

these inhibitors structurally resemble the substrate

An inhibitor may bind at an alternate site affectingcatalytic activity without affecting substrate binding

Many inhibitors do both

Most common typesCompetitive

Mixed or Non-competitive

Uncompetitive

I nhibitors act in a variety of mechanisms


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Competitive Inhibition

Competitive inhibitor competes with a substrate forthe enzyme - substrate binding site

Malonate is a

competitive

inhibitor of

succinate for

succinate

dehydrogenase


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A competitive inhibitor reduces the amount of

free enzyme available for substrate binding

thus increasing the Km for the substrate

The effect of a competitive inhibitor can be

overcome with high concentrations of the

substrate



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UnimolecularReaction

Bimolecular

Reaction



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Uncompetitive Inhibition

An uncompetitiveinhibitor binds tothe enzyme substratecomplex but not tofree enzyme

The result is adecrease in Vmaxand Km

The effect of an

uncompetitiveinhibitor can not beovercome by highconcentrations of thesubstrate


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Uncompetitive Inhibition


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Uncompetitive


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Mixed or Non-Competitive Inhibition

The inhibitor can bind to both free enzyme and the ES

complex

The affinity of the inhibitor to the two complexes might be

different

If binding of inhibitor changes the affinity for the substrate, Km will be

changed and called mixed inhibition

If only Vmax affected called Non-competitive inhibitor


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Mixed Inhibition


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The result will be decrease inVmax and either an increase ordecrease in Km

The effect of an non-competitiveinhibitor can only be partiallyovercome by high concentrationsof the substrate

Mixed Inhibition


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Non-Competitive


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kinetic enzymes

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