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All about Enzyme reaction Kinetics.

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Enzyme kinetics is the study of rates of Enzyme kinetics is the study of rates of

reactions catalyzed by enzymes.

d[S] d[P]

v = S P

k

v

d[S]

dt

d[P]

dt dt dt

The reaction rate (velocity, v) can be

described in several ways.

Disappearance of substrate, S

Appearance of product, P

These equations relate velocity to These equations relate velocity to

concentration of reactants and products.

Consider the reaction catalyzed by

triose phosphate isomerase.

As [GAP] decreases, the [DHAP]

increases increases.

GAP DHAP

Reaction velocity can be thought of

as concentration vs. time.

Many enzymes react with substrates

in a nonlinear fashion.

The shape here

is hyperbolic. is hyperbolic.

Sh i di t Shape indicates,

in part, that E and

S combine to

form an ES

complex

S S E + S ES

KEY CONCEPTS KEY CONCEPTS

Simple chemical reactions are described Simple chemical reactions are described

in terms of rate constants.

The Michaelis-Menten equation describes The Michaelis-Menten equation describes

enzyme-catalyzed reactions in terms of

K d V K

M

and V

max

.

Rate equations describe chemical

processes.

A unimolecular reaction has a velocity

(rate) that is dependent on the ( ) p

concentration of only one substrate.

v = A P

k

k [A] here k has nits of sec

1

v A P

v = k [A], where k has units of sec

-1

Rate equations describe chemical

processes.

A bimolecular (second order) reaction

has a velocity (rate) that is dependent on y ( ) p

two substrate concentrations.

v = A + B P

k

k [A] [B] (or k [A]

2

or k [B]

2

) v = k [A] [B] (or k [A]

2

or k [B]

2

)

where k has units of M

-1

sec

-1

Many enzymes obey Michaelis-Menten Many enzymes obey Michaelis Menten

kinetics.

E + S ES E + P

k

1

k

2

E + S ES E + P

k

-1

Rate limiting step Rate limiting step

d[P]

P bl

v

d[P]

dt

k2[ES]

Problem:

[ES] is difficult to measure!

dt

What can we do?

Try to re express the rate Try to re-express the rate.

k

k

E + S ES E + P

k

1

k

-1

k

2

-1

O i t th t h l

d[ES]

One point that helps:

k

1

[E] [S] - k

-1

[ES] - k

2

[ES]

d[ES]

dt

Depletion of ES

Formation

of ES

Assume steady state equilibrium Assume steady state equilibrium.

For most of the duration of the reaction, ,

[ES] remains steady as substrate is

converted to product converted to product.

d[ES] d[ES]

dt

0

Michaelis Menten Equation Michaelis-Menten Equation

k

k

E + S ES E + P

k

1

k

-1

k

2

d[P]

-1

v

d[P]

dt

k2[ES]

= k

1

[E] [S] - k

-1

[ES] - k

2

[ES]

d[ES]

dt

0

dt

Derivation of the

Michaelis-Menten Equation

= k

1

[E] [S] - k

-1

[ES] - k

2

[ES]

d[ES]

dt

0

dt

Thus: k [E] [S] = [ES] (k + k ) Thus: k

1

[E] [S] = [ES] (k

-1

+ k

2

)

[E]

total

= [ES] + [E] Since

[E] = [E]

total

- [ES]

Substitute

here

Derivation of the

Michaelis-Menten Equation

k

1

([E]

total

[S] - [ES] [S]) = [ES] (k

-1

+ k

2

)

Divide both sides by k

1

K

M

[E]

total

[S] - [ES] [S] = [ES] (k

-1

+ k

2

)

k

1

[E]

total

[S] - [ES] [S] = [ES] K

M

Derivation of the

Michaelis-Menten Equation

[E]

total

[S] - [ES] [S] = [ES] K

M

Rearrange:

[E]

total

[S] = [ES] (K

M

+ [S])

[ES] = [E] [S] [ES] = [E]

total

[S]

K

M

+ [S]

M

[S]

Derivation of the

Michaelis-Menten Equation

v

d[P]

k2[ES]

k [E] [S]

V

max

v

dt

k2[ES]

k

2

[E]

total

[S]

K

M

+ [S]

v =

M

[ ]

V

max

[S]

K [S]

v =

The Michaelis-

Menten Equation

K

M

+ [S]

Menten Equation

The Michaelis-Menten equation is

hyperbolic.

V is where the reaction velocity reaches its plateau V

max

is where the reaction velocity reaches its plateau

K

M

is the substrate

concentration at V concentration at V

max

KEY CONCEPTS: Section 7 2 KEY CONCEPTS: Section 7-2

The kinetic parameters K k and The kinetic parameters K

M

, k

cat

, and

k

cat

/K

M

are experimentally determined.

K

M

and V

max

values can be derived for

M max

enzymes that do not follow the Michaelis-

Menten model Menten model.

The catalytic rate constant determines y

how quickly an enzyme can act.

k =catalytic rate k

cat

= catalytic rate

constant, turnover

k

t

=k

2

k

cat

k

2

k

cat

= V

max

/[E]

total

k

t

/K

M

indicates catalytic efficiency k

cat

/K

M

indicates catalytic efficiency.

Wh t li it th t l ti f ? What limits the catalytic power of an enzyme?

Electronic rearrangements during formation of the

transition state

Frequency of productive enzyme collision with

substrate with the maximumbeing the diffusion- substrate, with the maximum being the diffusion

controlled limit

Enzymes reach catalytic perfection when their

t i diff i t ll d rate is diffusion-controlled.

The Lineweaver-Burk plot linearizes

Michaelis-Menten kinetics data.

V

max

[S]

v =

K

M

+ [S]

Take the reciprocal of both sides:

v

1

=

K

M

V

+

[S]

1

V

1

v

V

max

[S]

V

max

The Lineweaver-Burk plot linearizes

Michaelis-Menten kinetics data.

Notice how the data are weighted heavily

here due to the linearization!

Not all enzymes fit the Michaelis-

Menten model.

Some enzymes have multiple substrates.

Not all enzymes fit the Michaelis-

Menten model.

Some enzyme-catalyzed reactions have

many steps. y p

Not all enzymes fit the Michaelis-

Menten model.

With allosteric enzymes, binding of a substrate

at one active site can affect the catalytic activity y y

of other active sites.

Not all enzymes fit the Michaelis-

Menten model.

Allosteric enzymes exhibit cooperativity.

Velocity plot is sigmoidal!

KEY CONCEPTS: Section 7 3 KEY CONCEPTS: Section 7-3

Substances that bind irreversibly to an Substances that bind irreversibly to an

enzyme can inhibit its activity.

A competitive inhibitor appears to

increase K

M

without affecting V

max

.

M

g

max

Transition state analogs can act as

competitive inhibitors competitive inhibitors.

Noncompetitive, mixed, and p

uncompetitive inhibitors decrease k

cat

.

Allosteric regulators can inhibit or activate Allosteric regulators can inhibit or activate

enzymes.

Some inhibitors act irreversibly. y

A suicide inhibitor

Competitive inhibitors bind to the

same site as the substrate.

Competitive inhibitors increase K

M

,

but do not affect V

max.

Transition state analogs often make

b tt i hibit th b t t l better inhibitors than substrate analogs.

Transition State

Transition State Analog

Noncompetitive inhibitors appear to

decrease V

max

and K

M.

Allosteric regulation can inhibit or

enhance enzyme activity.

Consider the example:

Phosphoenolpyruvate inhibits PFK by p py y

an allosteric feedback mechanism.

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