Enzymes: The Catalysts of Life

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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 The induced-fit model In the past, the enzyme was seen as rigid, with the substrate fitting into the active site like a key in a lock (lock-and-key model) A more accurate view is the induced-fit model, in which substrate binding at the active site induces a conformational change in the shape of the enzyme

41 Figure 6-5

42 Video: Closure of hexokinase via induced fit

43 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

44 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

45 Three common mechanisms of substrate activation
Bond distortion, making it more susceptible to catalytic attack Proton transfer, which increases reactivity of substrate Electron transfer, resulting in temporary covalent bonds between enzyme, substrate

46 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

47 Figure 6-6

48 Figure 6-6A

49 Figure 6-6B

50 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

51 Figure 6-7

52 Enzyme Kinetics Enzyme kinetics describes the quantitative aspects of enzyme catalysis and the rate of substrate conversion into products Reaction rates are influenced by factors such as the concentrations of substrates, products, and inhibitors

53 Initial reaction rates
Initial reaction rates are measured over a brief time, during which the substrate concentration has not yet decreased enough to affect the rate of reaction

54 Most Enzymes Display Michaelis–Menten Kinetics
Initial reaction velocity (v), the rate of change in product concentration per unit time, depends on the substrate concentration [S] At low [S], doubling [S] will double v, but as [S] increases each additional increase in [S] results in a smaller increase in v When [S] becomes very large the value of v reaches a maximum

55 Figure 6-8

56 Vmax and saturation As [S] tends toward infinity, v approaches an upper limiting value, maximum velocity (Vmax) The value of Vmax can be increased by adding more enzyme The inability of increasingly higher substrate concentrations to increase the reaction velocity beyond a finite upper value is called saturation 56

57 The Michaelis–Menten Equation
Michaelis and Menten postulated a theory of enzyme action Enzyme E first reacts with the substrate, to form a transient complex, ES ES then undergoes the catalytic reaction to generate E and P 57

58 The Michaelis–Menten Equation (continued)
The above model, under steady state conditions gives the Michaelis–Menten equation Km (the Michaelis constant) = the concentration of substrate that gives half maximum velocity 58

59 What Is the Meaning of Vmax and Km?
We can understand the relationship between v and [S], and the meaning of Vmax and Km by considering three cases regarding [S] 59

60 Case 1: Very Low Substrate Concentration ([S] << Km)
If [S] << Km Then, Km + [S] = [Km] So at very low [S], the initial velocity of the reaction is roughly proportional to [S]

61 Case 2: Very High Substrate Concentration ([S] >> Km)
If [S] >> Km Then, Km + [S] = [S] So at very high [S], the initial velocity of the reaction is independent of variation in [S] and Vmax is the velocity at saturating substrate concentrations

62 Vmax Vmax is an upper limit determined by
The time required for the actual catalytic reaction How many enzyme molecules are present The only way to increase Vmax is to increase enzyme concentration

63 Figure 6-9

64 Case 3: ([S] = Km) If [S] is equal to Km [
This shows that Km is the specific substrate concentration at which the reaction proceeds at one half its maximum velocity

65 Why Are Km and Vmax Important to Cell Biologists?
The lower the Km value for a given enzyme and substrate, the lower the [S] range in which the enzyme is effective Vmax is important, as a measure of the potential maximum rate of the reaction By knowing Vmax, Km, and the in vivo substrate concentration, we can estimate the likely rate of the reaction under cellular conditions

66 Turnover number Vmax can be used to determine turnover number, kcat
kcat is the rate at which substrate molecules are converted to product by a single enzyme at maximum velocity Turnover numbers vary greatly among enzymes

67 Table 6-2

68 The Double-Reciprocal Plot Is a Useful Means of Linearizing Kinetic Data
Lineweaver and Burk inverted both sides of equation 6-7 to give This is known as the Lineweaver–Burk equation

69 The double-reciprocal plot
A plot of 1/v vs 1/[S] is called the double-reciprocal plot This linear plot takes the general form of y = mx + b, where m is the slope and b the y-intercept The slope is Km/Vmax, the y-intercept is 1/Vmax, and the x-intercept is –1/Km

70 Figure 6-10

71 The Eadie–Hofstee Plot
The Lineweaver–Burk equation has some limitations The Eadie–Hofstee equation is an alternative that allows Vmax to be determined from x-intercept and Km from slope

72 Figure 6-11

73 Determining Km and Vmax: An Example
Consider the following reaction, important in energy metabolism: glucose + ATP → glucose-6-phosphate + ADP To analyze this reaction, begin by determining initial velocity at several substrate concentrations For two substrates, they must be varied one at a time, with saturating levels of the other hexokinase

74 Figure 6-12

75 Determining Km and Vmax for hexokinase
For Figure 6-12, glucose is variable, wheras ATP is at saturating concentrations Each tube contains a known concentration of glucose between 0.05 and 0.40 mM In each tube, the reaction is initiated by adding a fixed amount of hexokinase The rate of product formation is determined

76 Reaction velocity relative to glucose concentration
The data points in Figure 6-12 give a hyperbolic curve Most kinetic data generated this way will fit a similar type of curve, illustrating the need for a linear representation of the results A double reciprocal plot makes determination of Km and Vmax easier

77 Figure 6-13

78 Enzyme Inhibitors Act Either Irreversibly or Reversibly
Enzymes are influenced (mostly inhibited) by products, alternative substrates, substrate analogs, drugs, toxins, and allosteric effectors The inhibition of enzyme activity plays a vital role as a control mechanism in cells Drugs and poisons frequently exert their effects by inhibition of specific enzymes

79 Inhibitors important to enzymologists
Inhibitors of greatest use to enzymologists are substrate analogs and transition state analogs These are compounds that resemble real substrates or transition states closely enough to occupy the active state but not closely enough to complete the reaction Substrate analogs are important tools in fighting infectious diseases

80 Reversible and irreversible inhibition
Irreversible inhibitors, which bind the enzyme covalently, cause permanent loss of catalytic activity and are generally toxic to cells For example, heavy metal ions, nerve gas poisons, some insecticides Reversible inhibitors bind enzymes noncovalently and can dissociate from the enzyme

81 Reversible inhibition (continued)
The fraction of enzyme available for use in a cell depends on the concentration of the inhibitor and how easily the enzyme and inhibitor can dissociate The two forms of reversible inhibitors are competitive inhibitors and noncompetitive inhibitors

82 Competitive inhibition
Competitive inhibitors bind the active site of an enzyme and so compete with substrate for the active site Enzyme activity is inhibited directly because active sites are bound to inhibitors, preventing the substrate from binding

83 Figure 6-14A

84 Noncompetitive inhibition
Noncompetitive inhibitors bind the enzyme molecule outside of the active site They inhibit activity indirectly by causing a conformation change in the enzyme that Inhibits substrate binding at the active site, or Reduces catalytic activity at the active site

85 Figure 6-14B

86 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

87 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

88 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

89 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

90 Figure 6-15

91 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

92 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

93 Figure 6-16A

94 Figure 6-16B

95 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

96 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

97 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. . 97

98 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 Protein kinases catalyze the phosphorylation of other proteins 98

99 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 99

100 Figure 6-17A

101 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 101

102 Figure 6-17B

103 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 103

104 Figure 6-18

105 RNA Molecules as Enzymes: Ribozymes
Some RNA molecules have been found to have catalytic activity; these are called ribozymes Self-splicing rRNA from Tetrahymena thermophila and ribonuclease P are examples It is thought by some that RNA catalysts predate protein catalysts, and even DNA