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Thermodynamics Research LaboratoryMapa InstituteofTechnology

ChungYuan Christian UniversitySchool ofChemical Engineering and Chemistry

ENZYMES I

Lecturer: Prof. Allan N. Soriano, Ph.D. Ch.E.

Email: [email protected]

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Outline

Introduction How Enzymes Work

Enzyme Kinetics:

- Mechanistic Models for Simple Enzyme Kinetics- Exper imentall y Determining Rate Parameters forMichaelis-M enten Type Kinetics

- Kinetic models for different inhibitions

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Introduction

ENZYMES are usually proteins of high molecular weight(15,000 < MW < several million daltons) that act as

CATALYSTS.

Recently, some RNA molecules had been shown to be

catalytic, but majority of cellular reactions are mediated byprotein catalysts.

-RNA molecules that have catalytic properties are called

ribozymes.

Enzymes arespecific, versatile, and very effective biologicalcatalysts, resulting in much higher reaction ratesas compared

to chemically catalyzed reactions under ambient conditions.

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Introduction (Cont.)

More than 2000 enzymes are known.

Enzymes are named by addingthe suffix

ase to the end of thesubstrate, such as urease, or the reaction catalyzed, such as

alcohol dehydrogenase.

Some enzymes have asimple structure, such as a folded

polypeptide chain (typical of most hydrolytic enzymes), butmany enzymes have more than one subunit.

Some protein enzymes require a nonprotein group for their

activity.

- Thisgroup is either a cofactor, such as metal ions, Mg, Zn, Mn,

Fe, or a coenzyme, such as a complex organic molecule, NAD,

FAD, CoA, or some vitamins.

- An enzyme containing a nonprotein group is called a

haloenzyme.

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An enzyme containing a nonprotein group is called a

holoenzyme.- Theprotein part of this enzyme is the apoenzyme (holoenzyme

= apoenzyme + cofactor).

Enzymes that occur in several different molecular forms, but

catalyze the same reaction, are called isozymes. Some enzymes aregrouped together to form enzyme complexes.

Enzymes aresubstrate specific and are classifiedaccording to

the reaction they catalyze.

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Enzyme Commission of the InternationalUnion of Biochemistry

Common namesaction description + -ase

suffix Numerical system

EC 2.7.4.4


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Some Useful

CommercialEnzyme

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How Enzymes Work

Models of catalysis:

Lock and Key vs. Induced Fit

Induced Fit Modelthe substrate still needs to fit into the enzyme like a

key, but instead of simply fitting into the keyhole,

some type of modification is induced in the

substrate, enzyme, or both.

The modification begins the process of the reaction.

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How Enzymes Work (Cont.)

Induced Fit

Model

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Effect of an enzyme on a reaction

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Endergonic (nonspontaneous)if the energy

level of the products is greater than that of the

reactants (energy is absorbed)

Exergonic (spontaneous)if the energy level

of the products is less than the reactants

(energy is released)

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A species has two possible fates in thetransition state:

it may lose energy and return to the reactant form,

orit may lose energy and move to the product form.

These two fates lead to two equilibria:

one of the equilibria involves the reactant (substrate)and the transition state

the other involves the product(s) and the transition state

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Enzyme assays: F ixed time and Kinetic Enzyme assayan experiment to determine

the catalytic activity of an enzyme.

Measure either the rate of disappearance of thesubstrate or the rate of appearance of a product

Fixed time assaymeasures the amount of

reaction in a fixed amount of time Kinetic assayyou monitor the progress of a

reaction continuously

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Enzyme Kinetics

Two general types of inhibition:

Competitive inhibition:another species

competes with the substrate to interact with

the active site on the enzyme

Noncompetitive inhibition:the other

species binds to some site other than the

active site

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Enzyme Kinetics (Cont.)

General reaction pathway:

k- rate constant

Simplification: k-2 is negligible

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Rate Determination

Saturation pointall the enzyme molecules

are part of an enzyme-substrate complex

The Michaelis constant,

measured in terms of

molarity, is a roughmeasure of the enzyme

substrate affinity.

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Rate Determination

Enzyme-substrate complexa tightly-bound

grouping of the enzyme and the substrate.

The limit occurs when all the enzyme moleculesare part of a complex so that there are no free

enzyme molecules available to accommodate the

additional substrate molecules.

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Mechanistic Models for Simple Enzyme Kinetics

Both the quasi-steady-state approximationand the

assumption ofrapid equi li br iumshare the same few

initial steps in deriving a rate expression for the

simplified mechanism, in which the rate of product

formation is ESP

2k

dt

dv

Where vis the rate of product formation or substrate consumption in

mol/I-s.

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The rate constant k2 is often denoted as kcat in the

biological literature. The rate of variation of the ES

complex is

ESESSE

ES

211 kkkdt

d

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Since the enzyme is not consumed, the conservation

equation on the enzyme yields

ESEE0

At this point, an assumption is requi red to achieve ananalytical solution.

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The Rapid Equilibrium Assumption

Used essentially byHenri andMichaelis and Menten.

The equilibrium coefficient can be use to express [ES]

in terms of [S].

The equilibrium constant is ES

SE

1

1'

k

kK

m

Since [E] = [E0][ES] if enzyme is conserved, then

SSE

S/

SEES

'

0

11

0

mKkk

Where Kmis the dissociation

constant of the ES complex.

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Then by substitution:

02

''

0

2

'

0

22

Ewhere

S

S

S

SE

S

SEES

P

kV

K

V

Kkv

K

kk

dt

dv

m

m

m

m

m

Here, Vmis the maximum forward velocity of the reaction

and Kmis often called the Michaelis-Menten constant.

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Vmchanges if more enzyme is added, but the addition

of more substrate has no influence on Vm.

A low value ofKmsuggests that the enzyme has a

high affinity for the substrate.

Also, Kmcorresponds to the substrate concentration,

giving the half-maximal reaction velocity.

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The Quasi-Steady-State Assumption

In many cases, the assumption of rapid equilibrium

following mass-action kinetics is not valid, although

the enzyme-substrate reaction still shows saturation-

type kinetics.

First proposed by G. E. Briggs andJ. B. S. Haldane.

Computer simulations of the actual time course have

shown that in a closed system the quasi-steady-statehypothesis holds after a brief transient if[S0] >> [E0].

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By applying the quasi-steady-state assumption, we

find

21

01

21

1

SESESE

ESkk

k

kk

k

Solving for [ES],

S

SEES

1

21

0

k

kk

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Then by substitution:

02121

1

21

02

2

Eand/where

S

S

S

SE

ESP

kVkkkK

K

V

k

kk

kv

kdt

dv

mm

m

m

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Experimentally Determining Rate Parameters

for Michaelis-Menten Type Kinetics

The determination of values forKmand Vmwith high

precision can be difficult.

Typically, experimental data are obtained from

ini tial-rate experiments.

- A batch reactor is charged with a known amount of substrate

[S0] and enzyme [E0].- The product (or substrate concentration) is plotted against

time.

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- The initial slope of this curve is estimated (i.e., v = d[P]/dtlt=0

= -d[S]/dtlt=0).

- This value ofv then depends on the values of [E0] and [S0] in

the charge to the reactor.

- Many such experiments can be used to generate many pairs

ofv and [S] data.

- These could be plotted but the accurate determination ofKm

from such a plot is very difficult.

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Lineweaver-Burk Plot

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Eadie-Hofstee Plot

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Hanes-Woolf Plot

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Kinetic models for different inhibitions

Competitive I nhibition

A competitive inhibitor enters the active site of an

enzyme and, thus, prevents the substrate fromentering.

An increase in the substrate concentration

overcomes this inhibition.

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A Lineweaver-Burk plot indicating competitive inhibition.

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Noncompetitive inhibition

Noncompetitive inhibitors do not enter the active

site but instead bind to some other region of the

enzyme.

This type of inhibitor reduces the turnover number

of the enzyme.

Unlike competitive inhibition, an increase in thesubstrate does not overcome noncompetitive

inhibition.

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A Lineweaver-Burk plot indicating noncompetitive inhibition.

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Th d i R h L bCh Y Ch i ti U i it

See you in TAIWAN!

END

ENZYMES I

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