ENZYMESA protein with catalytic properties due to its power of specific activation

Chemical reactions Chemical reactions need an initial input of

energy = THE ACTIVATION ENERGY During this part of the reaction the

molecules are said to be in a transition state.

Reaction pathway

Making reactions go faster Increasing the temperature make

molecules move faster Biological systems are very sensitive to

temperature changes. Enzymes can increase the rate of

reactions without increasing the temperature.

They do this by lowering the activation energy.

They create a new reaction pathway “a short cut”

An enzyme controlled pathway

Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.

Enzyme structure Enzymes are

proteins They have a

globular shape A complex 3-D

structure

Human pancreatic amylase

© Dr. Anjuman Begum

The active site One part of an

enzyme, the active site, is particularly important

The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily

© H.PELLETIER, M.R.SAWAYA ProNuC Database

Cofactors An additional non-

protein molecule that is needed by some enzymes to help the reaction

Tightly bound cofactors are called prosthetic groups

Cofactors that are bound and released easily are called coenzymes

Many vitamins are coenzymes

Nitrogenase enzyme with Fe, Mo and ADP cofactorsJmol from a RCSB PDB file © 2007 Steve Cook

H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REESSTRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS

IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

The substrate

The substrate of an enzyme are the reactants that are activated by the enzyme

Enzymes are specific to their substrates

The specificity is determined by the active site

The Lock and Key Hypothesis Fit between the substrate and the active site of

the enzyme is exact Like a key fits into a lock very precisely The key is analogous to the enzyme and the

substrate analogous to the lock. Temporary structure called the enzyme-substrate

complex formed Products have a different shape from the

substrate Once formed, they are released from the active

site Leaving it free to become attached to another

substrate

The Lock and Key Hypothesis

Enzyme may be used again

Enzyme-substrate complex

E

S

P

E

E

P

Reaction coordinate

The Lock and Key Hypothesis

This explains enzyme specificity This explains the loss of activity

when enzymes denature

The Induced Fit Hypothesis Some proteins can change their shape

(conformation) When a substrate combines with an

enzyme, it induces a change in the enzyme’s conformation

The active site is then moulded into a precise conformation

Making the chemical environment suitable for the reaction

The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

The Induced Fit Hypothesis

This explains the enzymes that can react with a range of substrates of similar types

Hexokinase (a) without (b) with glucose substratehttp://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

Understanding Km

The "kinetic activator constant" Km is a constant Km is a constant derived from rate

constants Km is an approximation of the

dissociation constant of E from S Small Km means tight binding; high Km

means weak binding

Understanding Vmax

The theoretical maximal velocity Vmax is a constant Vmax is the theoretical maximal rate of

the reaction - but it is NEVER achieved in reality

To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate

Vmax is asymptotically approached as substrate is increased

The turnover number

A measure of catalytic activity kcat, the turnover number, is the

number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate.

Values of kcat range from less than 1/sec to many millions per sec

Factors affecting Enzymes

substrate concentration pH temperature inhibitors

Substrate concentration: Non-enzymatic reactions

The increase in velocity is proportional to the substrate concentration

Reaction velocity

Substrate concentration

Substrate concentration: Enzymatic reactions

Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied.

If you alter the concentration of the enzyme then Vmax will change too.

Reaction velocity

Substrate concentration

Vmax

Enzymes and [S]

[S]

Init

ial re

act

ion r

ate

/

arb

itra

ry u

nit

s

As soon as a reaction begins, [S] begins to fall and so it is important that initial reaction rates are measured

Enzymes and [S]

[S]

Init

ial r

eact

ion

rate

/ ar

bitr

ary

unit

s

Enzymes and [S]

[S]

Init

ial r

eact

ion

rate

/ ar

bitr

ary

unit

s

Increasing [S] increases collision rate and increases reaction rate

Enzymes and [S]

[S]

Init

ial r

eact

ion

rate

/ ar

bitr

ary

unit

sAll active sites are occupied. Enzymes are working at maximum rate.

All active sites are not

occupied

Enzymes and [Substrate]

[S]

Init

ial r

eact

ion

rate

/ ar

bitr

ary

unit

sMaximum turnover

number or Vmax has been reached

Enzymes and [enzyme]

[Enzyme]

Init

ial r

eact

ion

rate

/ ar

bitr

ary

unit

s

Can we explain this in terms of the proportions of active sites occupied?

What factor is limiting here?

Enzymes and temperature: a tale of two effects

Temperature / oC

Collision rate of enzymes and substratesNumber of enzymes remaining undenatured

React

ion r

ate

/ a

rbit

rary

unit

s

Enzymes and temperature

Temperature / oC

Increasing kinetic energy

increases successful

collision rate

React

ion r

ate

/ a

rbit

rary

unit

s

Enzymes and temperature

Temperature / oC

Permanent disruption of

tertiary structure leads to loss of

active site shape, loss of binding efficiency and

activity

React

ion r

ate

/ a

rbit

rary

unit

s

Enzymes and temperature

Temperature / oC

Optimum temperature

React

ion r

ate

/ a

rbit

rary

unit

s

Enzymes and pH The precise shape of an enzyme

(and hence its active site) depends on the tertiary structure of the protein

Tertiary structure is held together by weak bonds (including hydrogen bonds) between R-groups (or ‘side-chains’)

Changing pH can cause these side chains to ionise resulting in the loss of H-bonding…

Enzymes and pH

pH

Rea

ctio

n ra

te /

arbi

trar

y un

its

Either side of the optimum pH, the gradual ionising of the side-chains (R-groups) results in loss of H-bonding, 3o structure, active site shape loss of binding efficiency and eventually enzyme activity

Optimum pH

Enzymes and pH

pH

Rea

ctio

n ra

te /

arbi

trar

y un

its

This loss of activity is only truly denaturation at extreme pH since between optimum and these extremes, the loss of activity is reversible

Optimum pH

Enzymes and pH

Enzymes and inhibitors Inhibitors are molecules that prevent

enzymes from reaching their maximum turnover numbers

Some inhibitors compete with the substrate for the active site

Some inhibitors affect the active site shape by binding to the enzyme elsewhere on the enzyme

Active site directed inhibition

Non-active site directed inhibition

Active site directed inhibition (Competitive) Inhibitor resembles the substrate

enough to bind to active site and so prevent the binding of the substrate: Substrat

e

Inhibitor

Enzyme

Active site directed inhibition (Competitive) Inhibitor resembles the substrate

enough to bind to active site and so prevent the binding of the substrate: Substrat

e

Enzyme/Inhibitor complex

Enzyme activity is

lost

Enzymes and competitive inhibition

[S]

Init

ial re

act

ion r

ate

/

arb

itra

ry u

nit

sAt low [S], the enzyme is more likely

to bind to the inhibitor and so activity is markedly reduced

Uninhibited

Inhibited

Enzymes and competitive inhibition

[S]

Init

ial re

act

ion r

ate

/

arb

itra

ry u

nit

sAs [S] rises, the enzyme is

increasingly likely to bind to the substrate and so activity increases

Uninhibited

Inhibited

Enzymes and competitive inhibition

[S]

Init

ial re

act

ion r

ate

/

arb

itra

ry u

nit

sAt high [S], the enzyme is very unlikely to bind to the inhibitor and so maximum

turnover is achieved

Uninhibited

Inhibited

Non-active site directed inhibition (Non-competitive) Inhibitor does not resemble the

substrate and binds to the enzyme disrupting the active site

Substrate

Inhibitor

Enzyme

Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site

Substrate

Enzyme

Active site is changed irreversibilit

y

Non-competitive inhibition

Non-competitive inhibition Inhibitor does not resemble the

substrate and binds to the enzyme disrupting the active site

Substrate

Enzyme

Activity is permanentl

y lost

Enzymes and non-competitive inhibition

[S]

Init

ial re

act

ion r

ate

/

arb

itra

ry u

nit

s

Can we explain this graph in terms of limiting factors in the parts of

the graph A and B?

A B

Applications of inhibitors

Negative feedback: end point or end product inhibition

Poisons snake bite, plant alkaloids and nerve gases.

Medicine antibiotics, sulphonamides, sedatives and stimulants