1. Key topics about enzyme function:
CHAPTER 6 Enzymes
Key topics about enzyme function:
Physiological significance of enzymes
Origin of catalytic power of enzymes
Chemical mechanisms of catalysis
Description of enzyme kinetics and inhibition

2. What are enzymes? Enzymes are catalysts
Increase reaction rates without being used up
Most enzymes are globular proteins
However, some RNA (ribozymes and ribosomal RNA) also catalyze reactions
Study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s
Study of enzymes has dominated biochemistry in the past and continues to do so

4. Why biocatalysis over inorganic catalysts?
Greater reaction specificity: avoids side products
Milder reaction conditions: conducive to conditions in cells
Higher reaction rates: in a biologically useful timeframe
Capacity for regulation: control of biological pathways
Metabolites have many potential pathways of decomposition
Enzymes make the desired one most favorable

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

6. Reaction Conditions Compatible with Life
pH ≈7

7. Six Classes of Enzymes: Defined by the Reactions Catalyzed
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 enzymecatalyzed reaction. The terms ∆G‡uncat and ∆G‡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.

8. Enzyme-Substrate Complex
Enzymes act by binding substrates
The noncovalent enzyme substrate complex is known as the Michaelis complex
Description of chemical interactions
Development of kinetic equations

9. 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, ∆G‡, for the S  P and P  S reactions are indicated. ∆G ’ ° is the overall standard free-energy change in the direction S  P.

10. Enzymes Decrease ΔG‡
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 enzymecatalyzed reaction. The terms ∆G‡uncat and ∆G‡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.

11. Rate Enhancement by Enzymes

12. How to Lower G
Enzymes organize reactive groups into close proximity and proper orientation
Uncatalyzed bimolecular reactions
two free reactants  single restricted transition state
conversion is entropically unfavorable
Uncatalyzed unimolecular reactions
flexible reactant  rigid transition state conversion is
entropically unfavorable for flexible reactants
Catalyzed reactions
Enzyme uses the binding energy of substrates to organize
the reactants to a fairly rigid ES complex
Entropy cost is paid during binding
Rigid reactant complex  transition state conversion is entropically OK

13. Support for the Proximity Model
The rate of anhydride formation from esters and carboxylates shows a strong dependence on proximity of two reactive groups (work by Thomas C. Bruice’s group).
FIGURE 6–7 Rate enhancement by entropy reduction. Shown here are reactions of an ester with a carboxylate group to form an anhydride. The R group is the same in each case. (a) For this bimolecular reaction, the rate constant k is second-order, with units of M-1s-1. (b) When the two reacting groups are in a single molecule, and thus have less freedom of motion, the reaction is much faster. For this unimolecular reaction, k has units of s-1. Dividing the rate constant for (b) by the rate constant for (a) gives a rate enhancement of about 105 M. (The enhancement has units of molarity because we are comparing a unimolecular and a bimolecular reaction.) Put another way, if the reactant in (b) were present at a concentration of 1 M, the reacting groups would behave as though they were present at a concentration of 105 M. Note that the reactant in (b) has freedom of rotation about three bonds (shown with curved arrows), but this still represents a substantial reduction of entropy over (a). If the bonds that rotate in (b) are constrained as in (c), the entropy is reduced further and the reaction exhibits a rate enhancement of 108 M relative to (a).

14. Illustration of TS Stabilization Idea: Imaginary Stickase
FIGURE 6–5 An imaginary enzyme (stickase) designed to catalyze breakage of a metal stick. (a) Before the stick is broken, it must first be bent (the transition state). In both stickase examples, magnetic interactions take the place of weak bonding interactions between enzyme and substrate. (b) A stickase with a magnet-lined pocket complementary in structure to the stick (the substrate) stabilizes the substrate. Bending is impeded by the magnetic attraction between stick and stickase. (c) An enzyme with a pocket complementary to the reaction transition state helps to destabilize the stick, contributing to catalysis of the reaction. The binding energy of the magnetic interactions compensates for the increase in free energy required to bend the stick. Reaction coordinate diagrams (right) show the energy consequences of complementarity to substrate versus complementarity to transition state (EP complexes are omitted). ∆GM, the difference between the transition-state energies of the uncatalyzed and catalyzed reactions, is contributed by the magnetic interactions between the stick and stickase. When the enzyme is complementary to the substrate (b), the ES complex is more stable and has less free energy in the ground state than substrate alone. The result is an increase in the activation energy.

15. Peptidoglycan and Lysozyme
Peptidoglycan is a polysaccharide found in many bacterial cell walls
Cleavage of the cell wall leads to the lysis of bacteria
Lysozyme is an antibacterial enzyme

16. Peptidoglycan and Lysozyme
FIGURE 6–27b Hen egg white lysozyme and the reaction it catalyzes. (b) Reaction catalyzed by hen egg white lysozyme. A segment of a peptidoglycan polymer is shown, with the lysozyme binding sites A through F shaded. The glycosidic C—O bond between sugar residues bound to sites D and E is cleaved, as indicated by the red arrow. The hydrolytic reaction is shown in the inset, with the fate of the oxygen in the H2O traced in red. Mur2Ac is N-acetylmuramic acid; GlcNAc, N-acetylglucosamine. RO— represents a lactyl (lactic acid) group; —NAc and AcN—, an N-acetyl
group (see key).

17. What is enzyme kinetics?
Kinetics is the study of the rate at which compounds react
Rate of enzymatic reaction is affected by:
enzyme
substrate
effectors
temperature

19. How to Do Kinetic Measurements
Experiment:
Mix enzyme + substrate
Record rate of substrate disappearance/product formation as a function of time (the velocity of reaction)
Plot initial velocity versus substrate concentration.
Change substrate concentration and repeat
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.

20. Effect of Substrate Concentration
Ideal rate:
Deviations due to:
limitation of measurements
substrate inhibition
substrate prep contains inhibitors
enzyme prep contains inhibitors

21. Effect of Substrate Concentration
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.)

22. At high [S] velocity does not depend on [S]
Saturation Kinetics:
At high [S] velocity does not depend on [S]
FIGURE 6-12 Dependence of initial velocity on substrate concentration. This graph shows the kinetic parameters that define the limits of the curve at high and low [S]. At low [S], Km >> [S] and the [S] term in the denominator of the Michaelis-Menten equation (Eqn 6-9) becomes insignificant. The equation simplifies to V0 = Vmax[S]/Km and V0 exhibits a linear dependence on [S], as observed here. At high [S], where [S] >> Km, the Km term in the denominator of the Michaelis-Menten equation becomes insignificant and the equation simplifies to V0 = Vmax; this is consistent with the plateau observed at high [S]. The Michaelis-Menten equation is therefore consistent with the observed dependence of V0 on [S], and the shape of the curve is defined by the terms Vmax/Km at low [S] and Vmax at high [S].

23. Determination of Kinetic Parameters
Nonlinear Michaelis-Menten plot should be used to calculate parameters Km and Vmax.
Linearized double-reciprocal plot is good for analysis of two-substrate data or inhibition.

24. Lineweaver-Burk Plot: Linearized, Double-Reciprocal
BOX 6-1 FIGURE 1 A double-reciprocal or Lineweaver-Burk plot.

25. Identify Constraints and Assumptions
Total enzyme concentration is constant
Mass balance equation for enzyme: ETot = [E] + [ES]
It is also implicitly assumed that: STot = [S] + [ES] ≈ [S]
Steady state assumption
What is the observed rate?
Rate of product formation

26. Carry out the algebra 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

27. Enzyme efficiency is limited by diffusion: kcat/Km
Can gain efficiency by having high velocity or affinity for substrate
Catalase vs. acetylcholinesterase

28. 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

29. Competitive Inhibition
Competes with substrate for binding
Binds active site
Does not affect catalysis
No change in Vmax; apparent increase in KM
Lineweaver-Burk: lines intersect at the y-axis

30. Competitive 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.

31. Competitive Inhibition
BOX 6-2 FIGURE 1 Competitive inhibition.

32. Uncompetitive Inhibition
Only binds to ES complex
Does not affect substrate binding
Inhibits catalytic function
Decrease in Vmax; apparent decrease in Km
No change in Km/Vmax
Lineweaver-Burk: lines are parallel

33. Uncompetitive 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.

34. Uncompetitive Inhibition
BOX 6-2 FIGURE 2 Uncompetitive inhibition.

35. Mixed Inhibition Binds enzyme with or without substrate
Binds to regulatory site
Inhibits both substrate binding and catalysis
Decrease in Vmax; apparent change in Km
Lineweaver-Burk: lines intersect left from the y-axis
Noncompetitive inhibitors are mixed inhibitors such that there is no change in Km
FIGURE 6–15c Mixed inhibitors bind at a separate site, but may bind to either E or ES.

36. Mixed 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.

37. Mixed Inhibition
BOX 6-2 FIGURE 3 Mixed inhibition.

38. Enzyme activity can be regulated
Regulation can be:
noncovalent modification
covalent modification
irreversible
reversible

39. Noncovalent Modification: Allosteric Regulators
The kinetics of allosteric regulators differ from Michaelis-Menten kinetics.
FIGURE 6–34 Substrate-activity curves for representative allosteric enzymes. Three examples of complex responses of allosteric enzymes to their modulators. (a) The sigmoid curve of a homotropic enzyme, in which the substrate also serves as a positive (stimulatory) modulator, or activator. Note the resemblance to the oxygen-saturation curve of hemoglobin (see Fig. 5–12). The sigmoidal curve is a hybrid curve in which the enzyme is present primarily in the relatively inactive T state at low substrate concentration, and primarily in the more active R state at high substrate concentration. The curves for the pure T and R states are plotted separately in color. ATCase exhibits a kinetic pattern similar to this. (b) The effects of several different concentrations of a positive modulator (+) or a negative modulator (-) on an allosteric enzyme in which K0.5 is altered without a change in Vmax. The central curve shows the substrate-activity relationship without a modulator. For ATCase, CTP is a negative modulator and ATP is a positive modulator.

40. Some Reversible Covalent Modifications
FIGURE 6–35 Some enzyme modification reactions.

41. Zymogens are activated by irreversible covalent modification
FIGURE 6–38 Activation of zymogens by proteolytic cleavage. Shown here is the formation of chymotrypsin and trypsin from their zymogens, chymotrypsinogen and trypsinogen. The bars represent the amino acid sequences of the polypeptide chains, with numbers indicating the positions of the residues (the amino-terminal residue is number 1). Residues at the termini of the polypeptide fragments generated by cleavage are indicated below the bars. Note that in the final active forms, some numbered residues are missing. Recall that the three polypeptide chains (A, B, and C) of chymotrypsin are linked by disulfide bonds (see Fig. 6–19).

42. The blood coagulation cascade uses irreversible covalent modification
FIGURE 6–40 The coagulation cascades. The interlinked intrinsic and extrinsic pathways leading to the cleavage of fibrinogen to form active fibrin are shown. Active serine proteases in the pathways are shown in blue. Green arrows denote activating steps, and red arrows indicate inhibitory processes.

43. Some enzymes use multiple types of regulation
FIGURE 6–42 Regulation of muscle glycogen phosphorylase activity by phosphorylation. The activity of glycogen phosphorylase in muscle is subjected to a multilevel system of regulation involving much more than the covalent modification (phosphorylation) shown in Figure 6–36. Allosteric regulation, and a regulatory cascade sensitive to hormonal status that acts on the enzymes involved in phosphorylation and dephosphorylation, also play important roles. The activity of both forms of the enzyme is allosterically regulated by an activator (AMP) and by inhibitors (glucose 6-phosphate and ATP) that bind to separate sites on the enzyme. The activities of phosphorylase kinase and phosphorylase phosphatase 1 (PP1) are also regulated by covalent modification, via a short pathway that responds to the hormones glucagon and epinephrine. One path leads to the phosphorylation of phosphorylase kinase and phosphoprotein phosphatase inhibitor 1 (PPI-1). The phosphorylated phosphorylase kinase is activated and in turn phosphorylates and activates glycogen phosphorylase. At the same time, the phosphorylated PPI-1 interacts with and inhibits PP1. PPI-1 also keeps itself active (phosphorylated) by inhibiting phosphoprotein phosphatase 2B (PP2B), the enzyme that dephosphorylates (inactivates) it. In this way, the equilibrium between the a and b forms of glycogen phosphorylase is shifted decisively toward the more active glycogen phosphorylase a. Note that the two forms of phosphorylase kinase are both activated to a degree by Ca2+ ion (not shown). This pathway is discussed in more detail in Chapters 14, 15, and 23.

44. Chapter 6: Summary In this chapter, we learned:
why nature needs enzyme catalysis
how enzymes can accelerate chemical reactions
how to perform and analyze kinetic studies
how to characterize enzyme inhibitors
how enzyme activity can be regulated