1. Lehninger Principles of Biochemistry
David L. Nelson and Michael M. Cox
Lehninger Principles of Biochemistry
Fourth Edition
Chapter 6:
Enzymes
Copyright © 2004 by W. H. Freeman & Company

2. Definitions
Substrate: Species that is acted upon by enzyme
Prosthetic group: Coenzyme or cofactor tightly (permanently) bound to enzyme
Holoenzyme: Enzyme + prosthetic group
Apoenzyme (apoprotein): Enzyme – prosthetic group
Active Site: Pocket within enzyme in which reaction occurs
Ground State: Starting point for forward/backward reactions; lowest ENERGY
Transition State: Top of energy “hill”; forward and reverse reactions are equally likely

5. Thermodynamics of Enzyme Catalyzed Reactions
G: Free energy change
G°: Standard free energy change
(T = 298K; P = 1atm; Concentration = 1M)
G°: Biochemical standard free energy change
(As above, but pH 7)
Relationship between free energy change and equilibrium constant K:
G° = -RT ln K
G‡ = activation energy

8. Kinetics
Overall rate of reaction is determined by the RATE LIMITING STEP (slowest step)
G° involves equilibrium, but
G‡ involves reaction rate
Rate equation (rate law):
1st order rate: (V) = k[S], where V and S are velocity and substrate concentration, respectively
2nd order rate: V = k[S1][S2]
Rate constant (k) is related to G‡ by
What is the mathematical relationship between G‡ and k?

10. How do they work?
Covalent interactions, though transient and short-lived, activate chemical groups for reaction
Non-covalent interactions: Formation of weak bonds between E and S release energy (GB)
Free energy from the quantity of these weak interactions is used to catalyze reactions and
Weak interactions are optimized by enzyme-transition state binding (not enzyme-substate)

12. What contributes to G‡? How are these issues overcome?
Reduction in Entropy
Binding energy used to hold substrate in place for reaction
Reduction in hydration shell
Binding between E and S compensate for bonds lost between E and water
Conformational change in substrate
Binding to TS optimizes interactions
Conformational change in enzyme
Change in protein shape IMPROVES/INCREASES interactions: INDUCED FIT

13. Reduction of entropy by enzyme

14. Types of Catalysis
Acid-base: Charged intermediates are stabilized

16. Types of Catalysis Acid-base: Charged intermediates are stabilized
Specific acid-base: Uses H+ or OH- from solution
General acid-base: H+ transfer from amino acid proton donor
Q: Which amino acids are most likely to act as general acids or bases?
Covalent: Formation of transient, covalently attached intermediate provides alternate route to products, lower in activation energy
Can be E-S or C-S (C = cofactor)

18. Types of Catalysis Acid-base: Charged intermediates are stabilized
Specific acid-base: Uses H+ or OH- from solution
General acid-base: H+ transfer from amino acid proton donor
Q: Which amino acids are most likely to act as general acids or bases?
Covalent: Formation of transient, covalently attached intermediate provides alternate route to products, lower in activation energy
Can be E-S or C-S (C = cofactor)
Metal ion
Ionic interactions stabilize E-S
Electron donation for REDOX

19. Enzyme Kinetics: Michaelis-Menten Kinetics

20. Enzyme Kinetics
Rate of reaction is sensitive to [E]

21. G. P. Royer1 and Sheldon E. Broedel, Jr
G. P. Royer1 and Sheldon E. Broedel, Jr.2 Buford Biomedical, Frederick, MD1 Athena Environmental Sciences, Inc., Baltimore, MD2

22. Rate of reaction is sensitive to [E]
As reaction proceeds, [S] decreases while [P] increases
In early part of reaction, rates are linear. We call this rate “initial velocity (v0)

23. If we plot v0 vs. [S], the rate plateaus at a maximum
If we plot v0 vs. [S], the rate plateaus at a maximum. This “maximum rate” is called vmax.

24. How can we study enzyme kinetics?
E = enzyme
S = substrate
ES = enzyme-substrate complex (Michaelis complex)
2 Simplifying Assumptions:
Step 1 is in equilibrium because k-1 >> k2
d[ES]/dt = 0

30. Bisubstrate Reactions
Some enzymatic reactions require more than one substrate and produce more than one product
Substrates are named A, B, C, D…in order in which they add to enzyme
Products are named P, Q, R, S… in order in which they leave the enzyme
The number of substrates and products involved in a reaction is indicated as Uni, Bi, Ter, Quad
Example: A reaction with 2 substrates and 2 products is called “Bi Bi”

31. Types of Bisubstrate Mechanisms I. Sequential Reactions
Ordered Reactions: Order of substrate addition is compulsory A B P Q k1
k-1 k2
k-2 k4
k-4 k5
k-5 k3 E EA
EAB
k-3
EPQ EQ E

32. Types of Bisubstrate Mechanisms I. Sequential Reactions
Random Reactions: Order of substrate addition is NOT compulsory

33. Types of Bisubstrate Mechanisms II. Ping Pong Reactions
Some products are released before all substrates are added E Q P B A
EA-FP F
FB-EQ

34. Enzyme Inhibition

35. Competitive Inhibition
Inhibitor (I) competes with S for binding to active site

36. Uncompetitive Inhibition

37. Mixed Inhibition

39. pH Dependence of Enzyme Activity

40. Chymotrypsin: An example of catalysis

49. Other Examples of Enzyme Catalytic Mechanisms
See Hexokinase, Lysozyme, Enolase

50. Enzyme Regulation
Allosterism: Binding of inhibitors or activators cause conformational changes, altering enzyme activity and kinetics

51. Feedback inhibition

53. Covalent Modification

54. Cleavage of Inactive Enzyme Precursor: Zymogens