1. Enzymes: The Catalysts of Life
Chapter 6
Enzymes: The Catalysts of Life

2. Enzymes: The Catalysts of Life
Enzyme catalysis: virtually all cellular processes or reactions are mediated by protein (sometimes RNA) catalysts called enzymes
The presence of the appropriate enzyme makes the difference between whether a reaction can take place and whether it will take place

3. Activation Energy and the Metastable State
Many thermodynamically feasible reactions in a cell that could occur do not proceed at any appreciable rate
For example, the hydrolysis of ATP has G = –7.3 kcal/mol
ATP + H2O ADP + Pi
However, ATP dissolved in water remains stable for several days

4. Before a Chemical Reaction Can Occur, the Activation Energy Barrier Must Be Overcome
Molecules that could react with one another often do not because they lack sufficient energy
Each reaction has a specific activation energy, EA
EA: the minimum amount of energy required before collisions between the reactants will give rise to products

5. Transition state
Reactants need to reach an intermediate chemical stage called the transition state
The transition state has a higher free energy than that of the initial reactants

6. Figure 6-1A

7. Activation energy barrier
The rate of a reaction is always proportional to the fraction of molecules with an energy equal to or greater than EA
The only molecules that are able to react at a given time are those with enough energy to exceed the activation energy barrier, EA

8. Figure 6-1B

9. The Metastable State Is a Result of the Activation Barrier
For most reactions at normal cell temperature, the activation energy is so high that few molecules can exceed the EA barrier
Reactants that are thermodynamically unstable, but lack sufficient EA, are said to be in a metastable state
Life depends on high EAs that prevent most reactions in the absence of catalysts

10. Catalysts Overcome the Activation Energy Barrier
The EA barrier must be overcome in order for needed reactions to occur
This can be achieved by either increasing the energy content of molecules or by lowering the EA requirement

11. Increasing the energy content of a system
The input of heat can increase the kinetic energy of the average molecule, ensuring that more molecules will be able to take part in a reaction
This is not useful in cells, however, which are isothermal
Isothermal: constant in temperature

12. Lowering activation energy
If reactants can be bound on a surface and brought close together, their interaction will be favored and the required EA will be reduced
A catalyst enhances the rate of a reaction by providing such a surface and effectively lowering EA
Catalysts themselves proceed through the reaction unaltered

13. Figure 6-1C

14. Figure 6-1D

15. Enzymes as Biological Catalysts
All catalysts share three basic properties
They increase reaction rates by lowering the EA required
They form transient, reversible complexes with substrate molecules
They change the rate at which equilibrium is achieved, not the position of the equilibrium
Organic catalysts are enzymes

16. Most Enzymes Are Proteins
Most enzymes are known to be proteins
However, recently, it has been discovered that some RNA molecules also have catalytic activity
These are called ribozymes and will be discussed later

17. The Active Site
Every enzyme contains a characteristic cluster of amino acids that forms the active site
This results from the three dimensional folding of the protein, and is where substrates bind and catalysis takes place
The active site is usually a groove or pocket that accommodates the intended substrate(s) with high affinity

18. Figure 6-2

19. Figure 6-2A

20. Figure 6-2B

21. Amino acids involved in the active site
Of the 20 different amino acids, only a few are involved in the active site
These are cys, his, ser, asp, glu, and lys
These can participe in binding the substrate and several serve as donors or acceptors of protons

22. Cofactors
Some enzymes contain nonprotein cofactors needed for catalytic activity, often because they function as electron acceptors
These are called prosthetic groups and are usually metal ions or small organic molecules called coenzymes
Coenzymes are derivatives of vitamins

23. Prosthetic groups
Prosthetic groups are located at the active site and are indispensable for enzyme activity
Each molecule of the enzyme catalase has a multimeric structure called a porphyrin ring to which a necessary iron atom is bound
The requirement for certain prosthetic groups on some enzymes explains our requirements for trace amounts of vitamins and minerals

24. Enzyme Specificity
Due to the shape and chemistry of the active site, enzymes have a very high substrate specificity
Inorganic catalysts are very nonspecific whereas similar reactions in biological systems generally have a much higher level of specificity

25. Figure 6-3

26. Group specificity
Some enzymes will accept a number of closely related substrates
Others accept any of an entire group of substrates sharing a common feature
This group specificity is most often seen in enzymes involved in degradation of polymers

27. Enzyme Diversity and Nomenclature
Thousands of different enzymes have been identified, with enormous diversity
Names have been given to enzymes based on substrate (protease, ribonuclease, amylase), or function (trypsin, catalase)
Under the Enzyme Commission (EC), enzymes are divided into six major classes based on general function

28. Six classes of enzymes Oxidoreductases Transferases Hydrolases Lysases
Isomerases
Ligases

29. Table 6-1

30. Sensitivity to Temperature
Enzymes are characterized by their sensitivity to temperature
This is not a concern in homeotherms, birds and mammals, that maintain a constant body temperature
However, many organisms function at their environmental temperature, which can vary widely

31. Enzyme activity and temperatures
At low temperatures, the rate of enzyme activity increases with temperature due to increased kinetic activity of enzyme and substrate molecules
However, beyond a certain point, further increases in temperature result in denaturation of the enzyme molecule and loss of enzyme activity

32. Optimal temperature
The temperature range over which an enzyme denatures varies among enzymes and organisms
The reaction rate of human enzymes is maximum at 37oC (the optimal temperature), the normal body temperature
Most enzymes of homeotherms are inactivated by temperatures above 50–55oC

33. Figure 6-4A

34. Ranges of heat sensitivity
Some enzymes are unusually sensitive and will denature at temperatures as low as 40oC
Some enzymes retain activity at unusually high temperatures, such as the enzymes of archaea that live in acidic hot springs
Enzymes of cryophilic (cold-loving) organisms such as Listeria bacteria can function at low temperatures, even under refrigeration

35. Sensitivity to pH
Most enzymes are active within a pH range of about 3–4 units
pH dependence is usually due to the presence of charged amino acids at the active site or on the substrate
pH changes affect the charge of such residues, and can disrupt ionic and hydrogen bonds

36. Figure 6-4B

37. Sensitivity to Other Factors
Enzymes are sensitive to factors such as molecules and ions that act as inhibitors or activators
Most enzymes are also sensitive to ionic strength of the environment
This affects hydrogen bonding and ionic interactions needed to maintain tertiary conformation

38. Substrate Binding, Activation, and Catalysis Occur at the Active Site
Because of the precise chemical fit between the active site of the enzyme and its substrates, enzymes are highly specific

39. Substrate Binding
Once at the active site, the substrate molecules are bound to the enzyme surface in the right orientation to facilitate the reaction
Substrate binding usually involves hydrogen bonds, ionic bonds, or both
Substrate binding is readily reversible

40. Figure 6-5

41. Conformational change
The induced conformational change brings needed amino acid side chains into the active site, even those that are not nearby
Sometimes these are not nearby unless the substrate is bound to the active site

42. Substrate Activation
The role of the active site is to recognize and bind the appropriate substrate and also to activate it by providing the right environment for catalysis
This is called substrate activation, which proceeds via several possible mechanisms

43. The Catalytic Event The sequence of events
1. The random collision of a substrate molecule with the active site results in it binding there
2. Substrate binding induces a conformational change that tightens the fit, facilitating the conversion of substrate into products

44. Figure 6-6

45. Figure 6-6A

46. Figure 6-6B

47. The Catalytic Event (continued)
The sequence of events
3. The products are then released from the active site
4. The enzyme molecule returns to the original conformation with the active site available for another molecule of substrate

48. Figure 6-7

49. Enzyme Regulation
Enzyme rates must be continuously adjusted to keep them tuned to the needs of the cell
Regulation that depends on interactions of substrates and products with an enzyme is called substrate-level regulation
Increases in substrate levels result in increased reaction rates, whereas increased product levels lead to lower rates

50. Allosteric regulation and covalent modification
Cells can turn enzymes on and off as needed by two mechanisms: allosteric regulation and covalent modification
Usually enzymes regulated this way catalyze the first step of a multi-step sequence
By regulating the first step of a process, cells are able to regulate the entire process

51. Allosteric Enzymes Are Regulated by Molecules Other than Reactants and Products
Allosteric regulation is the single most important control mechanism whereby the rates of enzymatic reactions are adjusted to meet the cell’s needs

52. Feedback Inhibition
It is not in the best interests of a cell for enzymatic reactions to proceed at the maximum rate
In feedback (or end-product) inhibition, the final product of an enzyme pathway negatively regulates an earlier step in the pathway

53. Figure 6-15

54. Allosteric Regulation
Allosteric enzymes have two conformations, one in which it has affinity for the substrate(s) and one in which it does not
Allosteric regulation makes use of this property by regulating the conformation of the enzyme
An allosteric effector regulates enzyme activity by binding and stabilizing one of the conformations

55. Allosteric regulation (continued)
An allosteric effector binds a site called an allosteric (or regulatory) site, distinct from the active site
The allosteric effector may be an activator or inhibitor, depending on its effect on the enzyme
Inhibitors shift the equilibrium between the two enzyme states to the low affinity form; activators favor the high affinity form

56. Figure 6-16A

57. Figure 6-16B

58. Allosteric enzymes
Most allosteric enzymes are large, multisubunit proteins with an active or allosteric site on each subunit
Active and allosteric sites are on different subunits, the catalytic and regulatory subunits, respectively
Binding of allosteric effectors alters the shape of both catalytic and regulatory subunits

59. Allosteric Enzymes Exhibit Cooperative Interactions Between Subunits
Many allosteric enzymes exhibit cooperativity
As multiple catalytic sites bind substrate molecules, the enzyme changes conformation, which alters affinity for the substrate
In positive cooperativity the conformation change increases affinity for substrate; in negative cooperativity, affinity for substrate is decreased

60. Enzymes Can Also Be Regulated by the Addition or Removal of Chemical Groups
Many enzymes are subject to covalent modification
Activity is regulated by addition or removal of groups, such as phosphate, methyl, acetyl groups, etc. . 60

61. Phosphorylation and Dephosphorylation
The reversible addition of phosphate groups is a common covalent modification
Phosphorylation occurs most commonly by transfer of a phosphate group from ATP to the hydroxyl group of Ser, Thr, or Tyr residues in a protein 61

62. Dephosphorylation
Dephosphorylation, the removal of phosphate groups from proteins, is catalyzed by protein phosphatases
Depending on the enzyme, phosphorylation may be associated with activation or inhibition of the enzyme
Fisher and Krebs won the Nobel prize for their work on glycogen phosphorylase 62

63. Figure 6-17A

64. Regulation of glycogen phosphorylase
Glycogen phosphorylase exists as two inter- convertible forms
An active, phosphorylated form (glycogen phosphorylase-a)
An inactive, non-phosphorylated form (glycogen phosphorylase-b)
The enzymes responsible
Phosphorylase kinase phosphorylates the enzyme
Phosphorylase phosphatase removes the phosphate 64

65. Figure 6-17B

66. Proteolytic Cleavage
The activation of a protein by a one-time, irreversible removal of part of the polypeptide chain is called proteolytic cleavage
Proteolytic enzymes of the pancreas, trypsin, chymotrypsin, and carboxypeptidase, are examples of enzymes synthesized in inactive form (as zymogens) and activated by cleavage as needed 66

67. Figure 6-18