Six classes of enzymes

Factors affecting enzyme activity

• Post-transcriptional modification/ Regulatory events

• pH

• Temperature

• Enzyme or Substrate concentration

• Cofactors

What do enzymes do?

A catalyst is a substancethat accelerates a chemicalreaction without itself undergoing any net change6

Dynamic Equilibria

• Consider: A B

• The rate of the forward reaction (kf) is

10-4sec-1

• The rate of the back reaction (kb) is 10-6sec-1

• K = [B]/[A] or kf/kb = 100

• At equilibrium, there is 100X B than A

• Enzymes accelerate these rates

How do enzymes work?

Fre

e en

ergy

Reaction coordinate showing a transition state and activation energy

Activation energy

• The starting point for a reaction is called the ground state, which reflects the contribution to the free energy of the system by the reactant

• Equilibrium between reactants (substrates; S) and products (P) reflects the difference in free energies of their ground states

• In the previous example, free energy of P is lower than that of S, so the free energy is negative and equilibrium favors products (look at the y-axis)

• Activation energy is the energy need to reach the transition state

The transition state is an energetic barrier

• A favorable equilibrium does not meant that a reaction occurs at a detectable rate

• The rate is dependent on crossing the energy barrier for alignment of reacting groups, formation of transient intermediates, bond rearrangements, etc.

• The molecules are raised to a higher energy level to overcome this level.

• The peak of the “hill” is called the transition state

Enzymes are catalysts

• Catalysts enhance reaction rates by lowering activation energies

• Intermediates can

be observed

• Do not affect

reaction equilibria

How do enzymes work?

Transition state vs. Ground State theory

As Pauling, among others, suggested is catalysis aresult of an enzyme having a higher affinity for thetransition state

Do enzymes accelerate catalysis by putting substrates in close proximity?

OR

Still to this day a topic of debate, but presently it seems to be a little of both

Enzymes have active sites

Active site have unique designs

The catalytic power of enzymes

• The ability of enzymes to catalyze reactions lies in– 1. Chemical reactions of many types occur

between substrates and enzyme function groups (amino acids, metal ions, cofactors). These reactions allow the rearrangement of covalent bonds during enzyme-mediated reactions

– 2. Binding energy

Binding energy is optimized for the transition state

• Some weak interactions are formed in the ES complex, but the full complement of interactions are only met in the transition state.

• The transition state exists as a brief point in time

• These interactions also provide specificity!

Affinity for the Transition state

E + S E + (S)*

E + S ES (ES)*

knon

kcat

Ks

KTS

KTS = [E][S]*/[ES]* = [(kcat/Km)/knon]-1

For Triosephosphate isomerase KTS = 10-12, and Km = 10-4

Thus, this enzyme binds the transition state eight orders of magnitudemore strongly than the substrate.

Substrate binding also leads to rate enhancement through entropy reduction

Recognition of transition state effects have led to developments in analogs and catalytic antibodies

Components of catalytic mechanisms

General acid-base catalysis Covalent catalysis Metal Ion catalysis (nucleophile,

electrophile)

Acid-Base Catalysis

Several amino acids are capable of acid-base catalysis

• Know

Approx.

pKa of

Amino

acids

Covalent catalysis

• A transient covalent bond is formed between enzyme and substrate

• Covalent complexes undergo regeneration to give back free enzyme

• Combo of

Acid-base and

Covalent

Metal Ion catalysis

• Ionic interactions assist in orientation of substrate and stabilize charged transition states

• Mediate oxidation-reduction reactions

• Nearly 1/3 of all enzymes are metalloenzymes

Examples of enzymatic reactions

• Chymotrypsin – covalent catalysis; general acid-base catalysis

• Hexokinase – induced fit

• Enolase – metal dependence

• Lysozyme – an unproven mechanism

Hexokinase catalytic mechanism illustrates an additional important

principle

• Catalyzes the conversion of glucose and ATP to G6P and ADP; the hydroxyl group at C6 of glucose is similar in reactivity to water, how does this enzyme discriminate?

Hexokinase undergoes a conformational change upon substrate binding

• Specific for glucose binding not water

Hexokinase a nice model for Koshland’s induced-fit mechanism

• When glucose is not present, the enzyme is in an open, inactive form with catalytic amino acids out of position.

• When glucose (not water), and ATP bind, the binding energy induces the conformational change to catalytically active form