Enzyme catalysis

Enzyme

A protein with catalytic properties due to its power of

specific activation

How do enzymes catalyze chemical

reactions?

Theoretical concept: transition state theory

Catalytic mechanisms:

• Acid-base catalysis

• Covalent catalysis

• Metal ion catalysis

• Electrostatic catalysis

• Catalysis through proximity and orientation effects

• Catalysis by preferential transition state binding

Example: Serine proteases

Transition state

Making reactions go faster

Increasing the temperature makes 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

Transition state theory

Transition state diagrams illustrate the path of a reaction

The transition state - the point of highest energy, i.e. the

most unstable situation

ΔG‡ is the difference of energy of the reactants and the

transition state (free energy of activation)

ΔG‡ determines the rate of the reaction

The reaction rate is proportional to e- ΔG‡/RT

R is the gas constant and T the absolute temperature (Eyring

equation)

greater the value of ΔG‡, the slower the reaction rate

Acceleration of a reaction rate can therefore be achieved by

lowering ΔG‡

Enzymes reduce ΔG‡

Catalysts lower the energy of activation of a chemical

reaction and hence accelerate the rate of reaction

The rate of a chemical reaction can be accelerated by up to

1017-fold

The rate acceleration is enzyme specific and depends on the

nature of the chemical reaction.

a catalyst does not change the equilibrium constant

between reactants and products

Acid-base catalysis

A reaction is acid-catalyzed if proton donation (a Brønsted

acid) lowers the free energy of activation and leads to an

acceleration of the reaction rate.

A reaction is base-catalyzed if proton abstraction (a

Brønsted base) lowers the free energy of activation and leads

to an acceleration of the reaction rate.

Reactions catalyzed by the concerted action of a proton

donation and abstraction are called acid-base catalyzed.

Acid or base catalysis of keto-enol tautomerism

RNAase A - example for an enzyme utilizing acid-

base catalysis

Function: Hydrolysis of RNA to component nucleotides

His12 abstracts a proton from the 2’-OH group.

This promotes the nucleophilic attack on the

phosphorus resulting in P-O bond cleavage.

His119 acts as a general acid by protonation of the

oxyanion leaving group. This results in the

formation of a 2’,3’-cyclic intermediate which can

be isolated under certain conditions. Water is not

admitted to the active site and drives essentially

the reversal of the initial process leading to

complete hydrolysis.

Covalent catalysis

Catalyst forms a transient covalent linkage with the substrate

leading to rate enhancement.

Can be divided into two phases:

1. Nucleophilic reaction between the catalyst and the substrate

to form a covalent bond

2. Withdrawal of electrons from the reaction center by the

electrophilic catalyst (e.g. hydrolysis of the Schiff base).

Note that in cases where the nucleophilicity is the rate-determining step, the

reaction rate tends to increase with the basicity (pK) of the catalyst.

Covalent catalysis

Top: uncatalyzed decarboxylation of acetoacetate

Bottom: primary amines as catalyst for the decarboxylation

of acetoacetate

Groups involved in covalent catalysis Among the amino acids, the following side chains can be used for

covalent catalysis:

• Serine: hydroxyl group (an example will follow later!)

• Cysteine: thiol group

• Histidine: imidazol ring

• Aspartate & glutamate: carboxyl group

• Lysine: amino group (Schiff base formation)

In addition, enzymes can utilize organic compounds, so called coenzymes, such as pyridoxal phosphate and thiamine pyrophosphate (vitamins!) for covalent catalysis.

Metal ion catalysis

One third of all enzymes require the presence of metal ions for activity!

1. Metalloenzymes - contain tightly bound metal ions, most

commonly transition metals such as iron, copper, manganese

and cobalt.

2. Metal-activated enzyme - bind metal ions loosely from

solution, most commonly alkali and alkaline earth metals such as

sodium, potassium, magnesium and calcium

Roles of metal ions in catalysis

• Bind to substrate in order to properly align it for catalysis

• Mediate oxidation-reduction processes (redox biochemistry)

through reversible interchange of the metal ion’s oxidation state.

• Stabilization/shielding of negative charges during the catalytic

process.

Metal ions promote nucleophilic catalysis

Metal-bound water exhibits a much lower pKa value

The resulting metalbound hydroxyl group can act as a potent

nucleophile

The essential zinc in the active site is bound by three

histidine side chains.

Water occupies the fourth binding site and is

polarized by the zinc atom.

The bound water deprotonates and the resulting

hydroxyl attacks carbon dioxide converting it to

carbonate.

Proximity and orientation effects

Binding of the substrate facilitates catalysis in three ways:

Proximity - effect is contributing a rate acceleration of ca.

5-fold

Orientation - Reaction rates are accelerated approx. 100-

fold by this effect

Freezing out motion - the enzyme restricts the

translational and vibrational freedom of the substrate an

„prepares“ it for a transition state like structure. The rate

enhancement achieved in the order of 107

The active site

For catalysis

The shape and the chemical environment inside the active

site permits a chemical reaction to proceed more easily

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

Types of Cofactors

Coenzyme: The non-protein component, loosely bound to

apoenzyme by non-covalent bond.

Examples : vitamins or compounds derived from vitamins.

Prosthetic group - The non-protein component, tightly

bound to the apoenzyme by covalent bonds.

Some enzymes require cofactors

To take over chemical reactions that cannot be performed by

amino acid side chains

Required in diet of organisms

Organic molecules can associate with enzyme as cosubstrate

(NAD+)

The structure and reaction of NAD+

NAD+ obligatory cofactor

In alcohol dehydrogenase reaction

NADH dissociates from the enzyme to be re-oxidized in an

independent reaction

Prosthetic groups

Permanently associated with enzymes

By covalent bonds

Example: heme is bound to proteins called cytochromes

Summary

Enzymes

Transition states

Activation energy

Acid-base catalysis

Covalent catalysis

Metal ion catalysis

Cofactors/coenzymes/cosubstrates/prosthetic groups

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

What happens at the active site?

Lock and key hypothesis

In the lock-and-key model of enzyme action:

-the active site has a rigid shape

-only substrates with the matching shape can fit

-the substrate is a key that fits the lock of the active site

This explains enzyme specificity.

This explains the loss of activity when enzymes denature

This is an older model, however, and does not work for all

enzymes.

Enzymes-how do they work?

Induced-fit hypothesis:

o When a substrate begins to bind to an enzyme, interactions

induce a conformational change in the enzyme

o Results in a change of the enzyme from a low catalytic form

to a high catalytic form

o Induced-fit hypothesis requires a flexible active site

(b)

Catalysis in the Enzyme’s Active Site

In an enzymatic reaction

The substrate binds to the active site

Held by weak interactions (hydrogen bonds)

Side chains (R) from amino acids catalyze the conversion of substrate

to product

Product leaves the active site

Enzyme is free to take another substrate molecule into its active site

Cycle happens very fast

Metabolic reactions are reversible

Enzyme can catalyze both forward and reverse reactions

Enzyme catalyzes the reaction in the direction of equilibrium

Use variety of mechanisms to lower the activation energy and speed

up a reaction

The catalytic cycle of an enzyme

Substrates

Products

Enzyme

Enzyme-substrate

complex

1 Substrates enter active site; enzyme

changes shape so its active site

embraces the substrates (induced fit).

2 Substrates held in

active site by weak

interactions, such as

hydrogen bonds and

ionic bonds.

3 Active site (and R groups of

its amino acids) can lower EA

and speed up a reaction by

• acting as a template for

substrate orientation,

• stressing the substrates

and stabilizing the

transition state,

• providing a favorable

microenvironment,

• participating directly in the

catalytic reaction.

4 Substrates are

Converted into

Products.

5 Products are

Released.

6 Active site

Is available for

two new substrate

molecule

Enzyme Inhibitors

Certain chemicals can inhibit the action of enzymes

Inhibitors

Attach to enzyme by covalent bonds

Usually irreversible

Enzyme Inhibitors Many enzyme inhibitors bind by weak bonds

In that case inhibition is reversible

Some reversible inhibitors resemble the normal substrate molecule

and compete for admission into the active site

Competitive inhibitors

Reduce the productivity of enzymes by blocking substrates from

entering the active site

Competitive inhibitors

Figure 8.19 (b) Competitive inhibition

A competitive

inhibitor mimics the

substrate, competing

for the active site.

Competitive

inhibitor

A substrate can

bind normally to the

active site of an

enzyme.

Substrate

Active site

Enzyme

(a) Normal binding

Noncompetitive inhibitors

Do not directly compete with the substrate

They impede enzymatic reactions by binding to another part of the

enzyme

Causing enzyme to change its shape

Renders the active site less effective at catalyzing the conversion of

substrates to product

Noncompetitive inhibitors

Figure 8.19

A noncompetitive

inhibitor binds to the

enzyme away from

the active site, altering

the conformation of

the enzyme so that its

active site no longer

functions.

Noncompetitive inhibitor

(c) Noncompetitive inhibition

Regulation of enzyme activity helps control metabolism

A cell’s metabolic pathways

Must be tightly regulated

Controlling where and when various enzymes are active

Can be done by switching on and off certain genes that encode specific

enzymes

Allosteric Activation and Inhibition

Many enzymes are allosterically regulated

Have two or more polypeptide chains or subunits

Each has its own active site

The entire complex oscillates between two conformational states:

catalytically active and inactive

Simplest case of allosteric regulation:

Activating or inhibiting regulatory molecule binds to a regulatory site

(located where subunits join)

Binding of activator stabilizes the conformation that has functional

active site

Binding of inhibitor stabilizes inactive form of the enzyme

Subunits fit together so that conformational change in one subunit is

transmitted to all others

Activator or inhibitor that binds to one site will affect the active sites

of all subunits

Stabilized inactive

form

Allosteric activater

stabilizes active from Allosteric enyzme

with four subunits Active site

(one of four)

Regulatory

site (one

of four)

Active form

Activator

Stabilized active form

Allosteric activater

stabilizes active form

Inhibitor Inactive form Non-

functional

active

site

(a) Allosteric activators and inhibitors. In the cell, activators and inhibitors

dissociate when at low concentrations. The enzyme can then oscillate again.

Oscillation

Figure 8.20

Other kind of allosteric activation:

If enzyme has multiple subunits, binding (induced fit) of the

substrate to one subunit can trigger conformational change in all

other subunits

Cooperativity

Amplifies the response of enzyme to substrates

Cooperativity

Figure 8.20

Binding of one substrate molecule to

active site of one subunit locks

all subunits in active conformation.

Substrate

Inactive form Stabilized active form

(b) Cooperativity: another type of allosteric activation. Note that the

inactive form shown on the left oscillates back and forth with the active

form when the active form is not stabilized by substrate.

Feedback Inhibition

In feedback inhibition

The end product of a metabolic pathway shuts down the pathway

Some cells use this pathway to sythesize one amino acid from another

Prevents the cell from wasting chemical resources

Factors affecting enzymes

All enzymes work best at only one particular temperature and pH: this is called the optimum.

Factors that affect the rate of a reaction include:

o substrate concentration

o pH

o enzyme concentration

o surface area

o pressure

o temperature

Different enzymes have different optimum temperatures and pH values.

Factors affecting enzymes

If the temperature and pH changes sufficiently beyond an

enzyme’s optimum, the shape of the enzyme irreversibly

changes.

This affects the shape of the active site and means that the

enzyme will no longer work.

When this happens the enzyme is denatured.

pH and reaction rate

pH also affects the rate of enzyme-substrate complexes

Most enzymes have an optimum pH of around 7 (neutral)

Substrate concentraton and reaction

rate

The rate of reaction increases as substrate concentration

increases (at constant enzyme concentration)

Maximum activity occurs when the enzyme is saturated

(when all enzymes are binding substrate)

Structure of enzymes

Apoenzyme and Holoenzyme

Apoenzyme is the enzyme without its non-protein moiety

and it is inactive.

Holoenzyme is an active enzyme with its non-protein

component.

Summary

Substrate

Lock-and-key hypothesis

Induced fit

Inhibitions

Competitive/noncompetitive inhibition

Factors affecting enzymes

Apoenzymes vs Holoenzyme