1. Biochemistry 林正輝 612 #5220 Chapter 13: Enzymes
Chapter 14: Mechanisms of enzyme action
Chapter 15: Enzyme regulation
Chapter 17: Metabolism- An overview
Chapter 18: Glycolysis
Chapter 19: The tricarboxylic acid cycle
Chapter 20: Electron transport & oxidative phosphorylation
Chapter 22: Gluconeogenesis, glycogen metabolism, and the pentose phosphate pathway
林正輝
612
#5220

2. Chapter 13 Enzymes – Kinetics and Specificity Biochemistry by
Reginald Garrett and Charles Grisham

3. What are enzymes, and what do they do?
Biological Catalysts
Increase the velocity of chemical reactions

4. What are enzymes, and what do they do?
Thousands of chemical reactions are proceeding very rapidly at any given instant within all living cells
Virtually all of these reactions are mediated by enzymes--proteins (and occasionally RNA) specialized to catalyze metabolic reactions
Most cells quickly oxidize glucose, producing carbon dioxide and water and releasing lots of energy:
C6H12O O2  6 CO H2O kJ of energy
It does not occur under just normal conditions
In living systems, enzymes are used to accelerate and control the rates of vitally important biochemical reactions

5. Figure 13.1 Reaction profile showing large DG‡ for glucose oxidation, free energy change of -2,870 kJ/mol; catalysts lower DG‡, thereby accelerating rate.

6. Enzymes are the agents of metabolic function
Enzymes form metabolic pathways by which
Nutrient molecules are degraded
Energy is released and converted into metabolically useful forms
Precursors are generated and transformed to create the literally thousands of distinctive biomolecules
Situated at key junctions of metabolic pathways are specialized regulatory enzymes capable of sensing the momentary metabolic needs the cell and adjusting their catalytic rates accordingly

7. Figure 13.2 The breakdown of glucose by glycolysis provides a prime example of a metabolic pathway. Ten enzymes mediate the reactions of glycolysis. Enzyme 4, fructose 1,6, biphosphate aldolase, catalyzes the C-C bond- breaking reaction in this pathway.

8. 13.1 – What Characteristic Features Define Enzymes?
Enzymes are remarkably versatile biochemical catalyst that have in common three distinctive features:
Catalytic power
Specificity
Regulation

9. Catalytic power
Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates!
Urease is a good example:
Catalyzed rate: 3x104/sec
Uncatalyzed rate: 3x10 -10/sec
Ratio is 1x1014 (catalytic power)

10. Specificity
Enzymes selectively recognize proper substances over other molecules
The substances upon which an enzyme acts are traditionally called substrates
Enzymes produce products in very high yields - often much greater than 95%

11. Figure 13.2 The breakdown of glucose by glycolysis provides a prime example of a metabolic pathway. Ten enzymes mediate the reactions of glycolysis. Enzyme 4, fructose 1,6, biphosphate aldolase, catalyzes the C-C bond- breaking reaction in this pathway.

12. Figure A 90% yield over 10 steps, for example, in a metabolic pathway, gives an overall yield of 35%. Therefore, yields in biological reactions must be substantially greater; otherwise, unwanted by-products would accumulate to unacceptable levels.

13. Specificity
The selective qualities of an enzyme are recognized as its specificity
Specificity is controlled by structure of enzyme
the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield
The specific site on the enzyme where substrate binds and catalysis occurs is called the active site

14. Regulation
Regulation of an enzyme activity is essential to the integration and regulation of metabolism
Because most enzymes are proteins, we can anticipate that the functional attributes of enzymes are due to the remarkable versatility found in protein structure
Enzyme regulation is achieved in a variety of ways, ranging from controls over the amount of enzyme protein produced by the cell to more rapid, reversible interactions of the enzyme with metabolic inhibitors and activators (chapter 15)

15. Nomenclature
Traditionally, enzymes often were named by adding the suffix -ase to the name of the substrate upon which they acted: Urease for the urea-hydrolyzing enzyme or phosphatase for enzymes hydrolyzing phosphoryl groups from organic phosphate compounds
Resemblance to their activity: protease for the proteolytic enzyme
Trypsin and pepsin
International Union of Biochemistry and Molecular Biology (IUBMB)
Enzymes Commission number: EC #.#.#.#

16. Nomenclature
A series of four number severe to specify a particular enzyme
First number is class (1-6)
Second number is subclass
Third number is sub-subclass
Fourth number is individual entry
For example, ATP:D-glucose-6-phosphotransferase (glucokinase) is listed as EC
ATP + D-glucose  ADP + D-glucose-6-phosphate
A phosphate group is transferred from ATP to C-6-OH group of glucose, so the enzyme is a transferase (class 2)
Transferring phosphorus-containing groups is subclass 7
An alcohol group (-OH) as an acceptor is sub-subclass 1
Entry 2

17. Classification of protein enzymes
Oxidoreductases catalyze oxidation-reduction reactions
Transferases catalyze transfer of functional groups from one molecule to another
Hydrolases catalyze hydrolysis reactions
Lyases catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement
Isomerases catalyze intramolecular rearrangement (isomerization reactions)
Ligases catalyze reactions in which two molecules are joined (formation of bonds)

19. EC 2. 7. 1. 1 hexokinase EC 2. 7. 1. 2 glucokinase EC 2. 7. 1
EC hexokinase EC glucokinase EC ketohexokinase EC fructokinase EC rhamnulokinase EC galactokinase . .
EC adenosylcobinamide kinase

20. Many enzymes require non-protein components called cofactors to aid in catalysis
Coenzymes: many essential vitamins are constituents of coenzyme
Cofactors: metal ions
metalloenzymes
Holoenzyme: apoenzyme (protien) + prosthetic group

22. Other Aspects of Enzymes
Mechanisms - to be covered in Chapter 14
Regulation - to be covered in Chapter 15
Coenzymes - to be covered in Chapter 17

23. 13.2 – Can the Rate of an Enzyme-Catalyzed Reaction Be Defined in a Mathematical Way?
Kinetics is concerned with the rates of chemical reactions
Enzyme kinetics addresses the biological roles of enzymatic catalyst and how they accomplish their remarkable feats
In enzyme kinetics, we seek to determine the maximum reaction velocity that the enzyme can attain and its binding affinities for substrates and inhibitors
These information can be exploited to control and manipulate the course of metabolic events

24. Chemical kinetics A  P (A  I  J  P) rate or velocity (v)
v = d[P] / dt or v = -d[A] / dt
The mathematical relationship between reaction rate and concentration of reactant(s) is the rate law
v = -d[A] / dt = k [A]
k is the proportional constant or rate constant (the unit of k is sec-1)

25. Chemical kinetics v = -d[A] / dt = k [A]
v is first-order with respect to A
The order of this reaction is a first-order reaction
molecularity of a reaction
The molecularity of this reaction equal 1 (unimolecular reaction)

26. Figure 13. 4 Plot of the course of a first-order reaction
Figure 13.4 Plot of the course of a first-order reaction. The half-time, t1/2, is the time for one-half of the starting amount of A to disappear.

27. Chemical kinetics A + B  P + Q
The molecularity of this reaction equal 2 (bimolecular reaction)
The rate or velocity (v)
v = -d[A] / dt = -d[B] / dt = d[P] / dt = d[Q] / dt
The rate law is
v = k [A] [B]
The order of this reaction is a second-order reaction
The rate constant k has the unit of M-1 sec-1)

28. The Transition State
Reaction coordinate: a generalized measure of the progress of the reaction
Free energy (G)
Standard state free energy (25℃, 1 atm, 1 M/each)
Transition state
The transition state represents an intermediate molecular state having a high free energy in the reaction.
Activation energy:
Barriers to chemical reactions occur because a reactant molecule must pass through a high-energy transition state to form products.
This free energy barrier is called the activation energy.

29. Figure 13.5 Energy diagram for a chemical reaction (A→P) and the effects of (a) raising the temperature from T1 to T2 or (b) adding a catalyst. Raising the temperature raises the average energy of A molecules, which increases the population of A molecules having energies equal to the activation energy for the reaction, thereby increasing the reaction rate. In contrast, the average free energy of A molecules remains the same in uncatalyzed versus catalyzed reactions (conducted at the same temperature). The effect of the catalyst is to lower the free energy of activation for the reaction.

30. Decreasing G‡ increase reaction rate
Two general ways may accelerate rates of chemical reactions
Raise the temperature
The reaction rate are doubled by a 10℃
Add catalysts
True catalysts participate in the reaction, but are unchanged by it. Therefore, they can continue to catalyze subsequent reactions.
Catalysts change the rates of reactions, but do not affect the equilibrium of a reaction.

31. Most biological catalysts are proteins called enzymes (E).
The substance acted on by an enzyme is called a substrate (S).
Enzymes accelerate reactions by lowering the free energy of activation
Enzymes do this by binding the transition state of the reaction better than the substrate
The mechanism of enzyme action in Chapter 14

32. 13.3 – What Equations Define the Kinetics of Enzyme-Catalyzed Reactions?
The Michaelis-Menten Equation
The Lineweaver-Burk double-reciprocal plot
Hanes-Woolf plot
Vmax [S]
v =
Km + [S]

33. Figure A plot of v versus [A] for the unimolecular chemical reaction, A→P, yields a straight line having a slope equal to k.

34. Figure Substrate saturation curve for an enzyme-catalyzed reaction. The amount of enzyme is constant, and the velocity of the reaction is determined at various substrate concentrations. The reaction rate, v, as a function of [S] is described by a rectangular hyperbola. At very high [S], v = Vmax. That is, the velocity is limited only by conditions (temperature, pH, ionic strength) and by the amount of enzyme present; v becomes independent of [S]. Such a condition is termed zero-order kinetics. Under zero-order conditions, velocity is directly dependent on [enzyme]. The H2O molecule provides a rough guide to scale. The substrate is bound at the active site of the enzyme.

35. The Michaelis-Menten Equation
Louis Michaelis and Maud Menten's theory
It assumes the formation of an enzyme-substrate complex (ES)
E S ES
At equilibrium
k-1 [ES] = k1 [E] [S]
And
Ks = = k1
k-1
[E] [S]
k-1
[ES] k1

36. The Michaelis-Menten Equation
E S ES E + P
The steady-state assumption
ES is formed rapidly from E + S as it disappears by dissociation to generate E + S and reaction to form E + P
d[ES] dt
That is; formation of ES = breakdown of ES
k1 [E] [S] = k-1[ES] + k2[ES] k1 k2
k-1
= 0

37. Figure 13.8 Time course for the consumption of substrate, the formation of product, and the establishment of a steady-state level of the enzyme-substrate [ES] complex for a typical enzyme obeying the Michaelis-Menten, Briggs-Haldane models for enzyme kinetics. The early stage of the time course is shown in greater magnification in the bottom graph.

38. The Michaelis-Menten Equation
k1 [E] [S] = k-1[ES] + k2[ES] = (k-1+ k2) [ES]
[ES] = ( ) [E] [S]
Km =
Km is Michaelis constant
Km [ES] = [E] [S] k1
k-1+ k2
k-1+ k2 k1

39. The Michaelis-Menten Equation
Km [ES] = [E] [S]
Total enzyme, [ET] = [E] + [ES]
[E] = [ET] – [ES]
Km [ES] = ([ET] – [ES]) [S] = [ET] [S] – [ES] [S]
Km [ES] + [ES] [S] = [ET] [S]
(Km + [S]) [ES] = [ET] [S]
[ES] =
[ET] [S]
Km + [S]

40. The Michaelis-Menten Equation
[ES] =
The rate of product formation is
v = k2 [ES]
v =
Vmax = k2 [ET] v =
[ET] [S]
Km + [S]
k2 [ET] [S]
Km + [S]
Vmax [S]
Km + [S]

41. Understanding Km
The Michaelis constant Km measures the substrate concentration at which the reaction rate is Vmax/2.
Associated with the affinity of enzyme for substrate
Small Km means tight binding; high Km means weak binding

42. v = Km + [S] When v = Vmax / 2 Vmax Vmax [S] 2 Km + [S]
Km + [S] = 2 [S]
[S] = Km
Vmax [S]
Km + [S] =

44. The theoretical maximal velocity
Understanding Vmax
The theoretical maximal velocity
Vmax is a constant
Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality
To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate
Vmax is asymptotically approached as substrate is increased

45. The dual nature of the Michaelis-Menten equation
Combination of 0-order and 1st-order kinetics
When S is low ([s] << Km), the equation for rate is 1st order in S
When S is high ([s] >>Km), the equation for rate is 0-order in S
The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S
The actual estimation of Vmax and consequently Km is only approximate from each graph

46. Vmax [S] v = Km + [S] Vmax v = [S] Km v = Vmax
When S is low ([s] << Km), Km + [S]=Km
When S is high ([s] >>Km), Km + [S]= [S]
Vmax
v =
[S] Km
v =
Vmax

47. A measure of catalytic activity
The turnover number
A measure of catalytic activity
kcat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate.
kcat is a measure of its maximal catalytic activity
If the M-M model fits, k2 = kcat = Vmax/Et
Values of kcat range from less than 1/sec to many millions per sec (Table 13.4)

49. The catalytic efficiency
Name for kcat/Km An estimate of "how perfect" the enzyme is
kcat/Km is an apparent second-order rate constant
v = (kcat/Km) [E] [S]
kcat/Km provides an index of the catalytic efficiency of an enzyme
kcat/Km = k1 k2 / (k-1 + k2)

51. Linear Plots of the Michaelis-Menten Equation
Lineweaver-Burk plot
Hanes-Woolf plot
Smaller and more consistent errors across the plot
Nonlinear Lineweaver-Burk or Hanes-Woolf plots are a property of regulatory enzymes (allosteric enzymes)

52. V =
Km + [S]
V Vmax [S]
Vmax [S]
Km + [S] =
Figure 13.9 The Lineweaver-Burk double-reciprocal plot, depicting extrapolations that allow the determination of the x- and y-intercepts and slope.

53. Figure A Hanes-Woolf plot of [S]/v versus [S], another straight-line rearrangement of the Michalelis-Menten equation.

54. Enzymatic activity is strongly influenced by pH
Figure 13.11
The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has slightly alkaline pH optimum, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimum near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with pK' near 6 is essential to its activity. Might it be a histidine residue within the active site?

55. Figure 13. 12 The effect of temperature on enzyme activity
Figure The effect of temperature on enzyme activity. The relative activity of an enzymatic reaction as a function of temperature. The decrease in the activity above 50°C is due to thermal denaturation.

56. 13.4 – What Can Be Learned from the Inhibition of Enzyme Activity?
Enzymes may be inhibited reversibly or irreversibly
Reversible inhibitors may bind at the active site (competitive) or at some other site (noncompetitive)
Enzymes may also be inhibited in an irreversible manner
Penicillin is an irreversible suicide inhibitor

57. Competitive inhibition

58. k1 kcat E + S ES E + P + I EI k-1 k3 k-3
A competitive inhibitor competes with substrate for the binding site. It changes the apparent km.
kcat [E]t [S]
kcat [E]t [S]
Vmax[S]
V = = =
app
app
km (1 + [I]/ KI) + [S]
km + [S]
km + [S]
[I]
app
km = km ( )
KI= k-3 / k3 KI

59. Figure Lineweaver-Burk plot of competitive inhibition, showing lines for no I, [I], and 2[I]. Note that when [S] is infinitely large (1/[S] = 0), Vmax is the same, whether I is present of not. In the presence of I, the negative

60. Noncompetitive inhibition

61. {kcat (1 + [I]/ KI)} [E]t [S] Vmax [S] V= = = km + [S] km + [S]
E + S ES E + P
I I
EI + S EIS
k-1 k3
k-3 k3
k-3 k1
k-1
app
app
kcat [E]t [S]
{kcat (1 + [I]/ KI)} [E]t [S]
Vmax [S]
V= = =
km + [S]
km + [S]
km + [S]
[I]
app
Vmax = Vmax ( ) KI

62. KI = KI’
Figure Lineweaver-Burk plot of pure noncompetitive inhibition. Note that I does not alter Km but that it decreases Vmax. In the presence of I, the y-intercept is equal to (1/Vmax)(1 + I/KI).

63. KI = KI’
Figure Lineweaver-Burk plot of mixed noncompetitive inhibition. Note that both intercepts and the slope change in the presence of I. (a) When KI is less than KI'; (b) when KI is greater than KI'.

64. Uncompetitive inhibitionk1
kcat
E + S ES E + P + I
EIS
k-1 k3
k-3

65. Figure 13. 17 Lineweaver-Burk plot of pure uncompetitive inhibition
Figure Lineweaver-Burk plot of pure uncompetitive inhibition. Note that I does not alter Km but that it decreases Vmax. In the presence of I, the y-intercept is equal to (1/Vmax)(1 + I/KI).

67. Irreversible inhibition
Irreversible inhibition occurs when substances combine covalently with enzymes so as to inactivate them irreversibly.
Suicide substrates are inhibitory substrate analogs designed, via normal catalytic actions of the enzyme, a very reactive group is generated. This reactive group then forms a covalent bond with a nearby functional group within the active site of the enzyme, thereby causing irreversible inhibition
Almost all irreversible enzyme inhibitors are toxic substances, either natural or synthetic. Such as penicillin

68. Figure 13.18
Penicillin is an irreversible inhibitor of the enzyme glycoprotein peptidase, which catalyzes an essential step in bacterial cell wall synthesis. Penicillin consists of a thiazolidine ring fused to a b-lactam ring to which a variable group R is attached. A reactive peptide bond in the b-lactam ring covalently attaches to a serine residue in the active site of the glycopeptide transpeptidase. (The conformation of penicillin around its reactive peptide bond resembles the transition state of the normal glycoprotein peptidase substrate.) The penicilloyl-enzyme complex is catalytically inactive. The bond between the enzyme and penicillin is indefinitely stable; that is, penicillin binding is irreversible.

69. 13.5 - What Is the Kinetic Behavior of Enzymes Catalyzing Bimolecular Reactions?
Enzymes often use two (or more) substrates
Bisubstrate reactions:
A + B P + Q
Reactions may be sequential or single-displacement reactions
E + A + B  AEB  PEQ  E + P + Q
And they can be random or ordered
Ping-pong or double-displacement reactions
enzyme

70. Figure 13. 19 Single-displacement bisubstrate mechanism
Figure Single-displacement bisubstrate mechanism. Double-reciprocal plots of the rates observed with different fixed concentrations of one substrate (B here) are graphed versus a series of concentrations of A. Note that, in these Lineweaver-Burk plots for single-displacement bisubstrate mechanisms, the lines intersect to the left of the 1/v axis.

71. The conversion of AEB to PEQ is the rate-limiting step in random, single-displacement reactions

72. Figure Random, single-displacement bisubstrate mechanisms where A does not affect B binding, and vice versa. Note that the lines intersect at the 1/[A] axis. (If [B] were varied in an experiment with several fixed concentrations of A, the lines would intersect at the 1/[B] axis in a 1/v versus 1/[B] plot.)

73. Figure The structures of creatine and creatine phosphate, guanidinium compounds that are important in muscle energy metabolism.

74. In an ordered, single-displacement reaction
Similar to 1st-order reaction

75. double-displacement (ping-pong) reactions

76. Figure Double-displacement (ping-pong) bisubstrate mechanisms are characterized by Lineweaver-Burk plots of parallel lines when double-reciprocal plots of the rates observed with different fixed concentrations of the second substrate, B, are graphed versus a series of concentrations of A.

77. Glutamate:aspartate aminotransferase
Figure Glutamate:aspartate aminotransferase, an enzyme conforming to a double-displacement bisubstrate mechanism. Glutamate:aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme. The pyridoxal serves as the -NH2 acceptor from glutamate to form pyridoxamine. Pyridoxamine is then the amino donor to oxaloacetate to form asparate and regenerate the pyridoxal coenzyme form. (The pyridoxamine: enzyme is the E' form.) 

78. 13.6 – How Can Enzymes Be So Specific?
“Lock and key” was the first explanation for specificity
“Induced fit” provides a more accurate description
Induced fit favors formation of the transition-state intermediate

79. Figure 13.24
A drawing, roughly to scale, of H2O, glycerol, glucose, and an idealized hexokinase molecule. Note the tow domains of structure in hexokinase (a), between which the active site is located. Binding of glucose induces a conformational change in hexokinase. The two domains close together, creating the catalytic site (b). The shaded area in (b) represents solvent inaccessible surface area in the active site cleft that results when the enzyme binds substrate.

80. 13.7 – Are All Enzymes Proteins?
Ribozymes
Segments of RNA that display enzyme activity in the absence of protein
Examples: RNase P and peptidyl transferase
Abzymes
Antibodies raised to bind the transition state of a reaction of interest

81. Figure RNA splicing in Tetrahymena rRNA maturation: (a) the guanosine-mediated reaction involved in the autocatalytic excision of the Tetrahymena rRNA intron, and (b) the overall splicing process. The cyclized intron is formed via nucleophilic attack of the 3'-OH on the phosphodiester bond that is 15 nucleotides from the 5'-GA end of the spliced-out intron. Cyclization frees a linear 15-mer with a 5'-GA end.

82. The Ribosome is a ribozymes
Figure (a) The 50S subunit from H. marismortui. (b) The aminoacyl-tRNA (yellow) and the peptidyl-tRNA (orange) in the peptidyl transferase active site.

83. Figure 13.27 The peptidyl transferase reaction.

84. Antibody molecules can have catalytic activity - Abzymes
Antigen: Transition state
1000X

85. 13.8 Is It Possible to Design An Enzyme to Catalyze Any Desired Reaction?
A known enzyme can be “engineered” by in vitro mutagenesis, replacing active site residues with new ones that might catalyze a desired reaction
Another approach attempts to design a totally new protein with the desired structure and activity
This latter approach often begins with studies “in silico” – i.e., computer modeling
Protein folding and stability issues make this approach more difficult
And the cellular environment may provide complications not apparent in the computer modeling

86. (limited reaction)
Figure cis-1,2-Dichloroethylene (DCE) is an industrial solvent that poses hazards to human health.
Site-directed mutations (F108L, I129L, and C248I) have enabled the conversion of a bacterial epoxide hydrolase to catalyze the chlorinated epoxide hydrolase reaction.