1. Enzymes
Function and Kinetics

2. Enzymes catalyze chemical reactions (thousands).
Most would not occur alone at physiological conditions.
Enzymes facilitate chemical reactions by increasing their rate.
They act as a catalyst, i.e., the enzyme is regenerated:
They are specific: they carry out specific reactions on specific substrates.

3. Thermodynamics vs. Kinetics
ΔG: the change in the Gibbs free energy for the reaction
ΔG = G(product) G(reactant)
A B
reactant product
If G(reactant) > G(product), ΔG is negative.
- ΔG is the driving force for the reaction.
The reaction is spontaneous and energy will be released.
ΔG is also related to the equilibrium constant (Keq): ΔGo = -RT ln Keq
ΔG for the reaction says nothing about how fast the reaction will go.

4. Kinetics (how fast?) G
Enzymes can increase rates by fold.

5. “Active Site” Region of the protein that: 1) binds substrate
2) catalyzes the chemical reaction
Example:
Carboxypeptidase A
--synthesized in the pancreas
--delivered to the small intestine lumen
--role in digestion of protein
Removes amino acids from the C-terminus of peptides
Peptide a.a COO-

6. A Substrate: H3N-Gly-Tyr-COO- +
(H2O)
Glycine Tyrosine
In water, no bond cleavage takes place.

7. Carboxypeptidase A
Active Site: Binding of Substrate

10. Enzyme Contains Zinc Ion

11. Active Site: Catalytic Mechanism

14. Whole protein involved
(induced fit)

15. Types of Enzyme Reactions
Oxidoreductases
--electrons, H or O atoms moved from one substrate to another one
(e.g, alcohol dehydrogenase; alcohol to aldehyde)
Transferases
--transfer of a chemical group from one molecule to another
(e.g., a kinase that moves a phosphate from ATP to a protein)
Hydrolases
--use water to make two products from a substrate
(e.g., carboxypeptidase A described above)
Lyases
--cleave –C-C-, –C-N- or C-O bonds, or sometimes make them (synthase)
Isomerases
--move a group or a double bond within the molecule
Ligases
--join atoms together using energy, usually from ATP (synthetases)

16. Coenzymes and Cofactors
Many enzymes just use their own amino acids for catalysis.
Others require nonpolypeptide coenzymes/cofactors for catalysis.
Cosubstrate: modified during reaction and leaves the active site.
Prosthetic group: tightly bound, regenerated after reaction.
Metal ions Cofactor

17. Enzyme Kinetics
(Rates in mathematical terms)

18. E + S E∙S P E + S E∙S P k1 k3 k2 k1 kcat k2
kcat is the catalytic rate constant
(for the rate-determining step)
Add enzyme to substrate;
measure substrate and product concentrations over time

19. Effect of [S] on Initial Velocity
S1 to S4: increasing [S]
v0 = initial velocity (change in [product] over time; d[P]/dt)

20. Initial Velocity vs. [S]
[Eo ] Km
v =
kcat
[S] +
Michaelis-Menten Equation

21. [Eo ] is total enzyme concentrationKm
v =
kcat
[S] +
[Eo ] is total enzyme concentration
kcat
[Eo ] = Vmax
(Maximum velocity at a given
[Eo] )
Km = k1
k2 + k3
If k3 << k2 , Km is the
dissociation constant (affinity)
E∙S
kcat and Km are constants!! Km
v =
[S] +
Vmax
If Km = [S],
v = 0.5 Vmax
Km is equivalent to the [S] that gives a velocity of one-half Vmax .

22. [S] >> Km
[S] ≈ Km
[S] << Km

23. Vmax = kcat [Eo]
Initial Velocities (Product vs. Time) for Different [Eo] at high [S]

24. How Fast Does an Enzyme Go, Actually?
Given by kcat in units of sec-1
kcat =
Vmax
[Eo ]
Also known as the turnover number: the number of substrate molecules converted to product per enzyme molecule per second

25. Determining Vmax and Km
Transform Data to a Lineweaver-Burk Plot:

26. Determining Vmax and Km
[S] +
Vmax
y = slope (x) y-intcp
Lineweaver-Burk Plot

27. Enzyme Inhibition

28. Enzyme Inhibition Competitive inhibition: Substrate Enzyme Enzyme
inhibitor
Substrate
Enzyme
Enzyme
-- inhibitor binds to part or all of the active site
-- blocks substrate binding.

29. Michaelis-Menten: Competitive Inhibition
Km (app)
Michaelis-Menten: Competitive Inhibition
(red: no inhibitor; green: inhibitor)
-- apparent Km higher: S must compete with I to fill 50% of active sites
-- Vmax not affected: S can outcompete I at high concentrations

31. Noncompetitive Inhibition:
Substrate
Enzyme
Substrate
Noncompetitive
Inhibitor
Enzyme
-- binds to an allosteric site
-- affects the active site
-- enzyme can’t catalyze the reaction
(ESI complex nonproductive) (lowers ES; decreases Vmax)
-- substrate still binds; Km not changed

32. Michaelis-Menten: Noncompetitive Inhibition
Vmax (app)
(red: no inhibitor; blue: inhibitor)
-- Km not affected: S does not compete with I at the active site
-- Vmax decreases (to Vmax (app))

34. ( ) ( ) Michaelis-Menten Equations for Inhibition Competitive: 1 +
1 +
[ I ] Ki
( ) Km
v =
[S] +
Vmax
= Km (app)
Noncompetitive:
1 +
[ I ] Ki +
( ) Km
v =
[S]
Vmax
= Vmax (app)
Ki is the EI dissociation constant

35. Enzyme Inhibitors in Medicine:
Examples