1. Six classes of enzymes

2. Factors affecting enzyme activity
Post-transcriptional modification/ Regulatory events pH
Temperature
Enzyme or Substrate concentration
Cofactors

3. What do enzymes do? A catalyst is a substance
that accelerates a chemical
reaction without itself
undergoing any net change 6

4. 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

5. How do enzymes work?
Free energy

6. Reaction coordinate showing a transition state and activation energy

7. 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

8. 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

9. Enzymes are catalysts
Catalysts enhance reaction rates by lowering activation energies
Intermediates can
be observed
Do not affect
reaction equilibria

10. How do enzymes work? Transition state vs. Ground State theory
Do enzymes accelerate catalysis by putting substrates
in close proximity? OR
As Pauling, among others, suggested is catalysis a
result of an enzyme having a higher affinity for the
transition state
Still to this day a topic of debate, but presently it seems to be
a little of both

11. Enzymes have active sites

12. Active site have unique designs

13. 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

15. 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!

16. 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 magnitude
more strongly than the substrate.

17. Substrate binding also leads to rate enhancement through entropy reduction

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

19. Components of catalytic mechanisms
General acid-base catalysis
Covalent catalysis
Metal Ion catalysis (nucleophile,
electrophile)

20. Acid-Base Catalysis

21. Several amino acids are capable of acid-base catalysis
Know
Approx.
pKa of
Amino
acids

22. 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 

23. 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

24. Examples of enzymatic reactions
Chymotrypsin – covalent catalysis; general acid-base catalysis
Hexokinase – induced fit
Enolase – metal dependence
Lysozyme – an unproven mechanism

32. 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?

33. Hexokinase undergoes a conformational change upon substrate binding
Specific for glucose binding not water

34. 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