6

6-1

Biochemistry by Mary K. Campbell

& Shawn O. Farrell

6

6-2

The Behavior

of Proteins:

Enzymes

6

6-3

Learning Objectives

1. What Makes Enzymes Such Effective Biological Catalysts?

2. What Is the Difference between the Kinetic and the

Thermodynamic Aspects of Reactions?

3. How Do Substrates Bind to Enzymes?

4. What are the features of the active site ?

5. What Are Some Examples of Enzyme-Catalyzed

reactions?

6. What Are Allosteric Enzymes ?

6

6-4

Enzyme Catalysis • Enzyme: a biological catalyst

• with the exception of some RNAs that catalyze their

own splicing , all enzymes are globular proteins.

• enzymes can increase the rate of a reaction by a

factor of up to 1020 over an uncatalyzed reaction

• some enzymes are so specific that they catalyze the

reaction of only one stereoisomer; others catalyze a

family of similar reactions

• The rate of a reaction depends on its activation

energy, DG°‡

• an enzyme provides an alternative pathway with a

lower activation energy (energy of activation)

6

6-5

Activation Energy Profile

An enzyme alters the rate of a reaction, but not its free

energy change or position of equilibrium

6

6-6

International Classifications of

Enzymes Type of reaction catalyzed Class No

.

Transfer of electrons (hydride ions or H atoms) Oxidoreductases 1

Group transfer reactions Transferases 2

Hydrolysis reactions (transfer of functional groups

to water) Hydrolases 3

Addition of groups to double bonds, or formation of

double bonds by removal of groups Lyases 4

Transfer of groups within molecules to yield

isomeric forms Isomerases 5

Formation of COC, COS, COO, and CON bonds by

condensation reactions coupled to ATP cleavage.

Ligases 6

6

6-7

Enzyme Catalysis Consider the reaction

H2 O2 H2 O + O2

No catalyst

Platinum surface

Catalase

75.2 18.0

48.9 11.7

23.0 5.5

Activation energy

(kJ/mol) (kcal/mol)

Relative

rate*

1

2.77 x 10 4

6.51 x 10 8

Reaction

Conditions

* Rates are given in arbitrary units relative to

a value of 1 for the uncatalyzed reaction at 37°C

6

6-8

Units of energy

A calorie (cal) is equivalent to the amount of heat

required to raise the temperature of 1 gram of

water from 14.5°C to 15.5°C.

A kilocalorie (kcal) is equal to 1000 cal.

A joule (J) is the amount of energy needed to

apply a 1-newton force over a distance of

1 meter.

A kilojoule (kJ) is equal to 1000 J.

1 kcal = 4.184 kJ

6

6-9

• In an enzyme-catalyzed reaction

• substrate, S: a reactant that is converted into

product by the enzyme

• active site: the small portion of the enzyme

surface where the substrate (s) becomes

bound.

E + S ES

enzyme-substrate

complex

6

6-10

How the Enzyme Works?

Enzymes are reusable!!!

6

6-11

Enzyme Catalysis • Two models have been developed to describe

formation of the enzyme-substrate complex

• lock-and-key model: substrate binds to the active

site of the enzyme with a complementarily in shape.

The active site is inflexible

• induced fit model: binding of the substrate induces a

change in the conformation of the enzyme that

results in a complementary fit

It assumes flexibility of the enzyme

The active site has a different 3D shape before

and after substrate binding

6

6-12

Lock and Key Model

6

6-13

Induced Fit Model

6

6-14

The Active Sites of Enzymes Have Some

Common Features

1. The active site is a three-dimensional cleft

formed by groups that come from different parts

of the amino acid sequence

2. The active site takes up a relatively small

part of the total volume of an enzyme.

3. Active sites are clefts or crevices.

6

6-15

4. Substrates are bound to enzymes by

multiple weak attractions

5. The specificity of binding depends on

the precisely defined arrangement of

atoms in an active site.

electrostatic interactions, hydrogen bonds,

van der Waals forces, and hydrophobic

interactions mediate reversible interactions

of biomolecules.

6

6-16

Binding of a substrate to an enzyme at the active

site.

The enzyme chymotrypsin, with bound substrate in

red

6

6-17

Chymotrypsin The Catalytic Triad. The catalytic triad,

shown on the left, converts serine 195

into a potent nucleophile, as

illustrated on the right.

6

6-18

Strategy and tactics. Chess and enzymes

have in common the use of strategy

6

6-19

Enzymes commonly employ one or more of the

following strategies to catalyze specific

reactions

1. Covalent catalysis:

In covalent catalysis, the active site

contains a reactive group, usually a

powerful nucleophile that becomes

temporarily covalently modified in the

course of catalysis. The proteolytic

enzyme chymotrypsin provides an

excellent example of this mechanism

6

6-20

2. General acid-base catalysis:

In general acid-base catalysis, a

molecule other than water plays the

role of a proton donor or acceptor.

Chymotrypsin uses a histidine

residue as a base catalyst to enhance

the nucleophilic power of serine

6

6-21

3. Metal ion catalysis. Metal ions can function

catalytically in several ways. For instance, a metal

ion may serve as an electrophilic catalyst,

stabilizing a negative charge on a reaction

intermediate. Alternatively, the metal ion may

generate a nucleophile by increasing the acidity of a

nearby molecule, such as water in the hydration of

CO2 by carbonic anhydrase

A nucleophile is a chemical species that donates an

electron pair to an electrophile to form a chemical bond

in relation to a reaction.

6

6-22

4. Catalysis by approximation:

Many reactions include two distinct

substrates. In such cases, the reaction

rate may be considerably enhanced by

bringing the two substrates together

along a single binding surface on an

enzyme.

6

6-23

Enzyme Catalysis: example • Chymotrypsin catalyzes the selective

hydrolysis of peptide bonds where the carboxyl

is contributed by Phe and Tyr

• it also catalyzes hydrolysis of the ester bond of

p-nitrophenyl esters

O2N OCCH3

O

+ H2 O

chymo-trypsin

O2N O-CH3 CO-+

pH > 7

p-Nitrophenylacetate

p- Nitrophenoxi de ion

O

6

6-24

Kinetics of Chymotrypsin Catalysis. Two

stages are evident in the cleaving of N-acetyl-l-

phenylalanine p-nitrophenyl ester by

chymotrypsin: a rapid burst phase (pre-steady

state) and a steady-state phase.

6

6-25

Note the hyperbolic

shape of the curve

6

6-26

6

6-27

Proteases Facilitating a Difficult Reaction

Protein turnover is an important process in living

systems . Proteins that have served their

purpose must be degraded so that their

constituent amino acids can be recycled for the

synthesis of new proteins. Proteins ingested in

the diet must be broken down into small

peptides and amino acids for absorption in the

gut. proteolytic reactions are important in

regulating the activity of certain enzymes and

other proteins

6

6-28

Proteolytic enzymes as an example. In vivo,

these enzymes catalyze proteolysis, the

hydrolysis of a peptide bond.

6

6-29

In the absence of a catalyst, the half-life

for the hydrolysis of a typical peptide

at neutral pH is estimated to be

between 10 and 1000 years.

Yet, peptide bonds must be hydrolyzed

within milliseconds in some

biochemical processes.

6

6-30

Most proteolytic enzymes also catalyze a

different but related reaction in vitro

namely, the hydrolysis of an ester bond.

6

6-31

Allosteric enzymes The activities of regulatory enzymes are

modulated in a variety of ways. Function

through reversible, noncovalent binding of

regulatory compounds called allosteric

modulators or allosteric effectors, which are

generally small metabolites or cofactors.

Other enzymes are regulated by reversible

covalent modification. Both classes of

regulatory enzymes tend to be multisubunit

proteins, and in some cases the regulatory

site(s) and the active site are on separate

subunits.

6

6-32

6

6-33

6

6-34

6

6-35

6

6-36

END

Chapter 6