1. Biochemistry
Lecture 8

2. Why Enzymes? Higher reaction rates Greater reaction specificity
Milder reaction conditions
Capacity for regulation
Metabolites have many potential pathways of decomposition
Enzymes make the desired one most favorable

3. Enzymatic Substrate Selectivity
No binding
Binding but no reaction
Example: Phenylalanine hydroxylase

4. FIGURE 6-2 Reaction coordinate diagram
FIGURE 6-2 Reaction coordinate diagram. The free energy of the system is plotted against the progress of the reaction S → P. A diagram of this kind is a description of the energy changes during the reaction, and the horizontal axis (reaction coordinate) reflects the progressive chemical changes (e.g., bond breakage or formation) as S is converted to P. The activation energies, ΔG˚, for the S → P and P → S reactions are indicated. ΔG′˚ is the overall standard free-energy change in the direction S → P.

5. FIGURE 6-3 Reaction coordinate diagram comparing enzyme-catalyzed and uncatalyzed reactions. In the reaction S → P, the ES and EP intermediates occupy minima in the energy progress curve of the enzyme-catalyzed reaction. The terms ΔG‡uncat and ΔG‡cat correspond to the activation energy for the uncatalyzed reaction and the overall activation energy for the catalyzed reaction, respectively. The activation energy is lower when the enzyme catalyzes the reaction.

6. Enzymes organizes reactive groups into proximity
How to Lower G?
Enzymes organizes reactive groups into proximity

7. Enzymes bind transition states best
How to Lower G?
Enzymes bind transition states best

8. How is TS Stabilization Achieved?
acid-base catalysis: give and take protons
covalent catalysis: change reaction paths
metal ion catalysis: use redox cofactors, pKa shifters
electrostatic catalysis: preferential interactions with TS
End result? Rate enhancements of 105 to 1017!

9. How is TS Stabilization Achieved?
covalent catalysis: change reaction paths

10. Enzyme Kinetics
Kinetics is the study of the rate at which compounds react
Rate of enzymatic reaction is affected by
Enzyme
Substrate
Effectors
Temperature

11. How to Do Kinetic Measurements

12. FIGURE 6-10 Initial velocities of enzyme-catalyzed reactions
FIGURE 6-10 Initial velocities of enzyme-catalyzed reactions. A theoretical enzyme catalyzes the reaction S ↔ P, and is present at a concentration sufficient to catalyze the reaction at a maximum velocity, Vmax, of 1 μM/min. The Michaelis constant, Km (explained in the text), is 0.5 μM. Progress curves are shown for substrate concentrations below, at, and above the Km. The rate of an enzyme-catalyzed reaction declines as substrate is converted to product. A tangent to each curve taken at time = 0 defines the initial velocity, V0, of each reaction.

13. FIGURE 6-11 Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction. The maximum velocity, Vmax, is extrapolated from the plot, because V0 approaches but never quite reaches Vmax. The substrate concentration at which V0 is half maximal is Km, the Michaelis constant. The concentration of enzyme in an experiment such as this is generally so low that [S] >> [E] even when [S] is described as low or relatively low. The units shown are typical for enzyme-catalyzed reactions and are given only to help illustrate the meaning of V0 and [S]. (Note that the curve describes part of a rectangular hyperbola, with one asymptote at Vmax. If the curve were continued below [S] = 0, it would approach a vertical asymptote at [S] = –Km.)

14. BOX 6-1 FIGURE 1 A double-reciprocal or Lineweaver-Burk plot.

15. What equation models this behavior?
Michaelis-Menten Equation

16. Meaning of Vmax and Km

17. Simple Enzyme Kinetics
The final form in case of a single substrate is
kcat (turnover number): how many substrate molecules can one enzyme molecule convert per second
Km (Michaelis constant): an approximate measure of substrate’s affinity for enzyme
Microscopic meaning of Km and kcat depends on the details of the mechanism

18. Two-substrate Reactions
Kinetic mechanism: the order of binding of substrates and release of products
When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms
Sequential mechanism
Ping-Pong (Double Displacement) mechanism

19. FIGURE 6-13 Common mechanisms for enzyme-catalyzed bisubstrate reactions. (a) The enzyme and both substrates come together to form a ternary complex. In ordered binding, substrate 1 must bind before substrate 2 can bind productively. In random binding, the substrates can bind in either order. (b) An enzyme-substrate complex forms, a product leaves the complex, the altered enzyme forms a second complex with another substrate molecule, and the second product leaves, regenerating the enzyme. Substrate 1 may transfer a functional group to the enzyme (to form the covalently modified E′), which is subsequently transferred to substrate 2. This is called a Ping-Pong or double-displacement mechanism.

20. Distinguishing Mechanism
Ping-Pong
Ternary Complex

21. Enzyme Inhibition
Inhibitors are compounds that decrease enzyme’s activity
Irreversible inhibitors (inactivators) react with the enzyme
one inhibitor molecule can permanently shut off one enzyme molecule
they are often powerful toxins but also may be used as drugs
Reversible inhibitors bind to, and can dissociate from the enzyme
- they are often structural analogs of substrates or products
- they are often used as drugs to slow down a specific enzyme
Reversible inhibitor can bind:
To the free enzyme and prevent the binding of the substrate
To the enzyme-substrate complex and prevent the reaction

22. FIGURE 6-15c Three types of reversible inhibition
FIGURE 6-15c Three types of reversible inhibition. (c) Mixed inhibitors bind at a separate site, but may bind to either E or ES.

23. BOX 6-2 FIGURE 3 Mixed inhibition.

24. FIGURE 6-15a Three types of reversible inhibition
FIGURE 6-15a Three types of reversible inhibition. (a) Competitive inhibitors bind to the enzyme's active site; KI is the equilibrium constant for inhibitor binding to E.

25. BOX 6-2 FIGURE 1 Competitive inhibition.

26. FIGURE 6-15b Three types of reversible inhibition
FIGURE 6-15b Three types of reversible inhibition. (b) Uncompetitive inhibitors bind at a separate site, but bind only to the ES complex; KI′ is the equilibrium constant for inhibitor binding to ES.

27. BOX 6-2 FIGURE 2 Uncompetitive inhibition.

28. FIGURE 6-31 Subunit interactions in an allosteric enzyme, and interactions with inhibitors and activators. In many allosteric enzymes the substrate binding site and the modulator binding site(s) are on different subunits, the catalytic (C) and regulatory (R) subunits, respectively. Binding of the positive (stimulatory) modulator (M) to its specific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change. This change renders the catalytic subunit active and capable of binding the substrate (S) with higher affinity. On dissociation of the modulator from the regulatory subunit, the enzyme reverts to its inactive or less active form.

29. FIGURE 6-34b Substrate-activity curves for representative allosteric enzymes. Three examples of complex responses of allosteric enzymes to their modulators. (b) The effects of a positive modulator (+) and a negative modulator (–) on an allosteric enzyme in which K0.5 is altered without a change in Vmax. The central curve shows the substrateactivity relationship without a modulator.

30. FIGURE 6-35 Some enzyme modification reactions.