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Biochemistry: A Short Course

First Edition

Biochemistry: A Short Course

First Edition

Tymoczko • Berg • Stryer

© 2010 W. H. Freeman and Company

CHAPTER 7

Mechanisms and Inhibitors

In this lesson we will investigate how In this lesson we will investigate how Catalytic strategies of enzymes works Catalytic strategies of enzymes works and then how enzyme activity and then how enzyme activity

can be can be modulated by environmental factors modulated by environmental factors distinct from allosteric signals distinct from allosteric signals

All enzymatic reactions go through aAll enzymatic reactions go through a transition state (unstable intermediate form with a structure between that of reactant and product).transition state (unstable intermediate form with a structure between that of reactant and product).

Reactants must collide precisely to form transition state. Reactants must collide precisely to form transition state.

Must haveMust have correct orientation.correct orientation. Must collide with enough energy = activation energy =Must collide with enough energy = activation energy = GG

Enzymes work byEnzymes work by loweringlowering GG Substrates are correctly oriented.Substrates are correctly oriented. All increase probability of reaction.All increase probability of reaction.

Transition states are stabilized.Transition states are stabilized. Another key aspect of enzymeAnother key aspect of enzyme catalyiscatalyis – –binding energy.binding energy. The fact that binding energy isThe fact that binding energy is maximalmaximal when the enzyme binds to the transition state favors the formation of the transition state and thereby promotes catalysis.when the enzyme binds to the transition state favors the formation of the transition state and thereby promotes catalysis.

Chemical CatalysisChemical Catalysis

Active site of most enzymes is lined with hydrophobic amino acids.Active site of most enzymes is lined with hydrophobic amino acids.

There are a few polar a.a. which make up the catalytic center of the active site and can be ionized.There are a few polar a.a. which make up the catalytic center of the active site and can be ionized. Histidine (basic a.a.) is common.Histidine (basic a.a.) is common.

Aspartate and glutamate - negatively chargedAspartate and glutamate - negatively charged Lysine and arginine - positively charged; electrostatic binding can occurLysine and arginine - positively charged; electrostatic binding can occur

2. General acid base catalysis2. General acid base catalysis

Enzymes that use this have a.a. side chains that can donate or accept electrons to substrate.Enzymes that use this have a.a. side chains that can donate or accept electrons to substrate.

Can accelerate a chemical reaction by a factor of 10-100.Can accelerate a chemical reaction by a factor of 10-100.

In general acid –base catalysts, a molecule other than water plays the role of a proton donor or acceptor.In general acid –base catalysts, 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. Chymotrypsin uses a histidine residue as a base catalyst to enhance the nucleophilic power of serine.

3. Metal ion catalysis3. Metal ion catalysis

Metal ion can function catalytically in several ways.Metal ion can function catalytically in several ways.

A.A.It serves as an electrophilic catalyst, stabilizing a negative charge on a reaction intermediate.It serves as an electrophilic catalyst, stabilizing a negative charge on a reaction intermediate.

B. B. Alternatively , the metal ion may generate a nucleophilic by increasing the acidity of a nearby molecule ,such as water. Alternatively , the metal ion may generate a nucleophilic by increasing the acidity of a nearby molecule ,such as water.

C.C.The metal ion may bind to the substrate , increasing the number of interactions with the enzyme and thus the binding energy.The metal ion may bind to the substrate , increasing the number of interactions with the enzyme and thus the binding energy.

Enzyme activity can be modulated by temperature , pH, and inhibitory molecules

Temperature Alters Enzymatic Activity

Tyrosinase

part of the pathway that

synthesizes the pigment

that results in dark fur,

has low tolerance for

heat. It inactive at normal

temperature but

functional at lower

temperature.

Heat sensitive

Tyrosinase Activity Curve

Why are Siamese markings only on their

extremities? cool enough for tyrosine to gain

function and produce pigment

[H

] Regulates Enzymatic Activity

Why do enzymes have a relatively narrow pH

optimum?

Protein Digestive Enzymes-

Pepsin stomach

Chymotrypsin

intestine

Activity of most

enzymes display a

bell shaped curve

Optimum

pH

varies

acid

ic

basic

If the pH is lowered , the - coo- If the pH is lowered , the - coo-

group will converted into –COOH so less group will converted into –COOH so less

enzyme activity enzyme activity

If the pH is raised the –NH3 + group If the pH is raised the –NH3 + group

loses an H+ to OH - , becoming a neutral loses an H+ to OH - , becoming a neutral

**- NH2 group , and the enzyme activity

• NH2 group , and the enzyme activity**

diminished. diminished.

The pH dependence of enzymes is due to The pH dependence of enzymes is due to

the presence of ionizable R groups the presence of ionizable R groups

Graphical representation of competitive Graphical representation of competitive

inhibitors: inhibitors:

affects K affects K

m m

(increases K (increases K

m m

--> decreases --> decreases

affinity; need more substrate to reach half- affinity; need more substrate to reach half-

saturation of enzyme) saturation of enzyme)

V

V

maxmax

unaffected unaffected

Substrate can out Substrate can out

compete inhibitor → compete inhibitor → V

V

maxmax

unchanged since unchanged since V

V

max max

k k

2 2

[E]

[E]

As concentration of a As concentration of a

competitive inhibitor competitive inhibitor

increases, higher increases, higher

concentrations of substrate concentrations of substrate

are required to attain a are required to attain a

particular reaction particular reaction

velocity. The reaction velocity. The reaction

pathway suggests how pathway suggests how

sufficiently high sufficiently high

Reaction

Pathway

Competitive Enzyme Inhibition page 95

Reaction Pathway

Substrate can out compete

Substrate can out compete

inhibitor → V

inhibitor → V

max max

unchanged

unchanged

since V

since V

maxmax

k

k

22

[E]

[E]

TT

Inhibitor binds in the

Inhibitor binds in the

active site →

active site →

K

K

M M

increases

increases

since

since

K

K

MM

k

k

-1-

k

k

22

k

k

11

e.g.

e.g.

AZT inhibition

AZT inhibition

of HIV reverse

of HIV reverse

transcriptaseactual

transcriptaseactual

substrate is dTTP

substrate is dTTP

deoxythymidine

deoxythymidine

Noncompetitive Enzyme Inhibition

The inhibitor and

The inhibitor and

substrate can bind

substrate can bind

simultaneously to an

simultaneously to an

enzyme molecule at

enzyme molecule at

different binding sites

different binding sites

Reaction Pathway

Noncompetitive Enzyme Inhibition

Reaction Pathway

Taking Taking enzyme out of enzyme out of

circulation

circulation →

→ V

V

max max

lowered lowered since since V V

max max

=

k

k

2 2

[E]

[E]

Inhibitor binds both E Inhibitor binds both E

and ES

and ES →

→ K

K

M M

unchanged unchanged

since

since K

K

M M

= (

= ( k

k

k

k

2 2

)/

)/ k

k

1 1