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Prentice Hall c2002Chapter 51 Principles of Biochemistry Fourth Edition Chapter 5 Properties of Enzymes Copyright © 2006 Pearson Prentice Hall, Inc. Horton.

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Presentation on theme: "Prentice Hall c2002Chapter 51 Principles of Biochemistry Fourth Edition Chapter 5 Properties of Enzymes Copyright © 2006 Pearson Prentice Hall, Inc. Horton."— Presentation transcript:

1 Prentice Hall c2002Chapter 51 Principles of Biochemistry Fourth Edition Chapter 5 Properties of Enzymes Copyright © 2006 Pearson Prentice Hall, Inc. Horton Moran Scrimgeour Perry Rawn

2 Prentice Hall c2002Chapter 52 Chapter 5 - Properties of Enzymes Catalyst - speeds up attainment of reaction equilibrium Enzymatic reactions - 10 3 to 10 17 faster than the corresponding uncatalyzed reactions Substrates - highly specific reactants for enzymes

3 Prentice Hall c2002Chapter 53 Properties of enzymes (continued) Stereospecificity - many enzymes act upon only one stereoisomer of a substrate Reaction specificity - enzyme product yields are essentially 100% (there is no formation of wasteful byproducts) Active site - where enzyme reactions take place

4 Prentice Hall c2002Chapter 54

5 Prentice Hall c2002Chapter 55 5.1 The Six Classes of Enzymes 1. Oxidoreductases (dehydrogenases) Catalyze oxidation-reduction reactions

6 Prentice Hall c2002Chapter 56 2. Transferases Catalyze group transfer reactions

7 Prentice Hall c2002Chapter 57 3. Hydrolases Catalyze hydrolysis reactions where water is the acceptor of the transferred group

8 Prentice Hall c2002Chapter 58 4. Lyases Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination (Synthases catalyze the addition to a double bond, the reverse reaction of a lyase)

9 Prentice Hall c2002Chapter 59 5. Isomerases Catalyze isomerization reactions

10 Prentice Hall c2002Chapter 510 6. Ligases (synthetases) Catalyze ligation, or joining of two substrates Require chemical energy (e.g. ATP)

11 Prentice Hall c2002Chapter 511 5.2 Kinetic Experiments Reveal Enzyme Properties A. Chemical Kinetics Experiments examine the amount of product (P) formed per unit of time (  [P] /  t) Velocity (v) - the rate of a reaction (varies with reactant concentration) Rate constant (k) - indicates the speed or efficiency of a reaction

12 Prentice Hall c2002Chapter 512 First order rate equation Rate for nonenzymatic conversion of substrate (S) to product (P) in a first order reaction: (k is expressed in reciprocal time units (s -1 ))  [P] /  t = v = k[S]

13 Prentice Hall c2002Chapter 513 Second order reaction For reactions: S 1 + S 2 P 1 + P 2 Rate is determined by the concentration of both substrates Rate equation: v = k[S 1 ] 1 [S 2 ] 1

14 Prentice Hall c2002Chapter 514 Pseudo first order reaction If the concentration of one reactant is so high that it remains essentially constant, reaction becomes zero order with respect to that reactant Overall reaction is then pseudo first-order v = k[S 1 ] 1 [S 2 ] 0 = k’[S 1 ] 1

15 Prentice Hall c2002Chapter 515 B. Enzyme Kinetics When [S] >> [E], every enzyme binds a molecule of substrate (enzyme is saturated with substrate) Under these conditions the rate depends only upon [E], and the reaction is pseudo-first order Enzyme-substrate complex (ES) - complex formed when specific substrates fit into the enzyme active site E + S ESE + P

16 Prentice Hall c2002Chapter 516 Fig 5.1 Effect of enzyme concentration [E] on velocity (v) Fixed, saturating [S] Pseudo-first order enzyme-catalyzed reaction

17 Prentice Hall c2002Chapter 517 Initial velocity (v o ) Velocity at the beginning of an enzyme-catalyzed reaction is v o (initial velocity) k 1 and k -1 represent rapid noncovalent association /dissociation of substrate from enzyme active site k 2 = rate constant for formation of product from ES E + SESE + P k1k1 k2k2 k -1

18 Prentice Hall c2002Chapter 518 Fig 5.2 Progress curve for an enzyme-catalyzed reaction The initial velocity (v o ) is the slope of the initial linear portion of the curve Rate of the reaction doubles when twice as much enzyme is used

19 Prentice Hall c2002Chapter 519 5.3 The Michaelis-Menten Equation Maximum velocity (V max ) is reached when an enzyme is saturated with substrate (high [S]) At high [S] the reaction rate is independent of [S] (zero order with respect to S) At low [S] reaction is first order with respect to S The shape of a v o versus [S] curve is a rectangular hyperbola, indicating saturation of the enzyme active site as [S] increases

20 Prentice Hall c2002Chapter 520 Fig 5.3 Plots of initial velocity (v o ) versus [S] (a) Each v o vs [S] point is from one kinetic run (b) Michaelis constant (K m ) equals the concentration of substrate needed for 1/2 maximum velocity

21 Prentice Hall c2002Chapter 521 The Michaelis-Menten equation Equation describes v o versus [S] plots K m is the Michaelis constant V max [S] v o = K m + [S]

22 Prentice Hall c2002Chapter 522 A. Derivation of the Michaelis-Menten Equation Derived from (1) Steady-state conditions: Rate of ES formation = Rate of ES decomposition (2) Michaelis constant: K m = (k -1 + k 2 ) / k 1 (3) Velocity of an enzyme-catalyzed reaction (depends upon rate of conversion of ES to E + P) v o = k 2 [ES]

23 Prentice Hall c2002Chapter 523 B. The Meanings of K m K m = [S] when v o = 1/2 Vmax K m  k -1 / k 1 = K s (the enzyme-substrate dissociation constant) when k cat << either k 1 or k -1 The lower the value of K m, the tighter the substrate binding K m can be a measure of the affinity of E for S

24 Prentice Hall c2002Chapter 524 Fig 5.4 K m and physiological substrate concentrations K m values for enzymes are typically just above [S], so that the enzyme rate is sensitive to small changes in [S]

25 Prentice Hall c2002Chapter 525 5.4 Kinetic Constants Indicate Enzyme Activity and Specificity Catalytic constant (k cat ) - first order rate constant for conversion of ES complex to E + P k cat most easily measured when the enzyme is saturated with S (region A on Figure 5) Ratio k cat /K m is a second order rate constant for E + S E + P at low [S] concentrations (region B on Figure 5)

26 Prentice Hall c2002Chapter 526 Fig 5.5 Meanings of k cat and k cat /K m

27 Prentice Hall c2002Chapter 527 Table 5.1 Examples of catalytic constants Enzymek cat (s -1 )

28 Prentice Hall c2002Chapter 528 Values of k cat /K m k cat /K m can approach rate of encounter of two uncharged molecules in solution (10 8 to 10 9 M -1 s -1 ) k cat /K m is also a measure of enzyme specificity for different substrates (specificity constant) rate acceleration = k cat /k n (k n = rate constant in the absence of enzyme)

29 Prentice Hall c2002Chapter 529

30 Prentice Hall c2002Chapter 530 5.5 Measurement of K m and V max Fig 5.6 The double- reciprocal Lineweaver- Burk plot is a linear transformation of the Michaelis-Menten plot (1/v o versus 1/[S])

31 Prentice Hall c2002Chapter 531 5.6 Kinetics of Multisubstrate Reactions Fig 5.7 Notations for bisubstrate reactions (a) Sequential (ordered or random) Ordered Random

32 Prentice Hall c2002Chapter 532 Fig. 5.7 (b) Bisubstrate reactions

33 Prentice Hall c2002Chapter 533 5.7 Reversible Enzyme Inhibition Inhibitor (I) binds to an enzyme and prevents formation of ES complex or breakdown to E + P Inhibition constant (K i ) is a dissociation constant EIE + I There are three basic types of inhibition: Competitive, Uncompetitive and Noncompetitive These can be distinguished experimentally by their effects on the enzyme kinetic patterns

34 Prentice Hall c2002Chapter 534 Fig 5.8 Reversible enzyme inhibitors (a) Competitive. S and I bind to same site on E (b) Nonclassical competitive. Binding of S at active site prevents binding of I at separate site. Binding of I at separate site prevents S binding at active site.

35 Prentice Hall c2002Chapter 535 Fig 5.8 (cont) (c) Uncompetitive. I binds only to ES (inactivates E) (d) Noncompetitive. I binds to either E or ES to inactivate the enzyme

36 Prentice Hall c2002Chapter 536 A. Competitive Inhibition Inhibitor binds only to free enzyme (E) not (ES) Substrate cannot bind when I is bound at active site (S and I “compete” for the enzyme active site) V max is the same with or without I (high S can still saturate the enzyme even in the presence of I) Apparent K m (K m app ) measured in the presence of I is larger than K M (measured in absence of I) Competitive inhibitors usually resemble the substrate

37 Prentice Hall c2002Chapter 537 Fig 5.9 Competitive inhibition. (a) Kinetic scheme. (b) Lineweaver-Burk plot

38 Prentice Hall c2002Chapter 538 Fig 5.10 Benzamidine competes with arginine for binding to trypsin

39 Prentice Hall c2002Chapter 539 B. Uncompetitive Inhibition Uncompetitive inhibitors bind to ES not to free E V max decreased by conversion of some E to ESI K m (K m app ) is also decreased Lines on double-reciprocal plots are parallel This type of inhibition usually only occurs in multisubstrate reactions

40 Prentice Hall c2002Chapter 540 Fig 5.11 Uncompetitive inhibition

41 Prentice Hall c2002Chapter 541 C. Noncompetitive Inhibition Noncompetitive inhibitors bind to both E and ES Inhibitors do not bind at the same site as S V max decreases K m does not change Inhibition cannot be overcome by addition of S Lines on double-reciprocal plot intersect on x axis

42 Prentice Hall c2002Chapter 542 Fig 5.12 Noncompetitive inhibition

43 Prentice Hall c2002Chapter 543 D. Uses of Enzyme Inhibition Fig 5.13 Comparison of a substrate (a) and a designed inhibitor (b) for the enzyme purine nucleoside phosphorylase

44 Prentice Hall c2002Chapter 544 5.8 Irreversible Enzyme Inhibition Irreversible inhibitors form stable covalent bonds with the enzyme (e.g. alkylation or acylation of an active site side chain) There are many naturally-occurring and synthetic irreversible inhibitors These inhibitors can be used to identify the amino acid residues at enzyme active sites Incubation of I with enzyme results in loss of activity

45 Prentice Hall c2002Chapter 545 Fig 5.14 Covalent complex with lysine residues Reduction of a Schiff base forms a stable substituted enzyme

46 Prentice Hall c2002Chapter 546 Inhibition of serine protease with DFP Diisopropyl fluorophosphate (DFP) is an organic phosphate that inactivates serine proteases DFP reacts with the active site serine (Ser-195) of chymotrypsin to form DFP-chymotrypsin Such organophosphorous inhibitors are used as insecticides or for enzyme research These inhibitors are toxic because they inhibit acetylcholinesterase (a serine protease that hydrolyzes the neurotransmitter acetylcholine)

47 Prentice Hall c2002Chapter 547 Fig 5.15 Reaction of DFP with Ser-195 of chymotrypsin

48 Prentice Hall c2002Chapter 548 Affinity labels for studying enzyme active sites Affinity labels are active-site directed reagents They are irreversible inhibitors Affinity labels resemble substrates, but contain reactive groups to interact covalently with the enzyme

49 Prentice Hall c2002Chapter 549 Fig 5.16 Irreversible inhibition of triose phosphate isomerase Bromo analog reacts with active site glutamate, and reduction with NaBH 4 stabilizes the adduct

50 Prentice Hall c2002Chapter 550 5.9 Site-Directed Mutagenesis Modifies Enzymes Site-directed mutagenesis (SDM) can be used to test the functions of individual amino acid side chains One amino acid is replaced by another using molecular biology techniques Bacterial cells can be used to synthesize the modified protein

51 Prentice Hall c2002Chapter 551 Practical applications of SDM SDM is also used to change the properties of enzymes to make them more useful Subtilisin protease was made more resistant to chemical oxidation by replacing Met-222 with Ala-222 (the modified subtilisin is used in detergents) A bacterial protease was made more heat stable by replacing 8 of 319 amino acids

52 Prentice Hall c2002Chapter 552 5.10 Regulation of Enzyme Activity Regulatory enzymes - activity can be reversibly modulated by effectors Such enzymes are usually found at the first unique step in a metabolic pathway (the first “committed” step) Regulation at this step conserves material and energy and prevents accumulation of intermediates

53 Prentice Hall c2002Chapter 553 Two Methods of regulation (1) Noncovalent allosteric regulation (2) Covalent modification Allosteric enzymes have a second regulatory site (allosteric site) distinct from the active site Allosteric inhibitors or activators bind to this site and regulate enzyme activity via conformational changes

54 Prentice Hall c2002Chapter 554 A. Phosphofructokinase-1 (PFK-1) Is an Allosteric Enzyme PFK-1catalyzes an early step in glycolysis (an ATP-generating pathway for glucose degradation) Phosphoenol pyruvate (PEP), an intermediate near the end of the pathway is an allosteric inhibitor of PFK-1 ADP is an allosteric activator of PFK-1

55 Prentice Hall c2002Chapter 555 Regulation of glycolysis by PFK-1 When the ratio [PEP]/[ADP] is high, flux through glycolysis decreases at the PFK-1 step When the ratio of [PEP]/[ADP] is low, PFK-1 is activated and glycolysis produces more ATP from ADP Thus the concentrations of PEP and ADP act allosterically through PFK-1 to regulate the activity of the entire pathway

56 Prentice Hall c2002Chapter 556 Fig 5.17 Reaction catalyzed by PFK-1

57 Prentice Hall c2002Chapter 557 Fig 5.18 Phosphoenolpyruvate (PEP)

58 Prentice Hall c2002Chapter 558 Effect of PEP and ADP on PFK-1 Both PEP and ADP affect the binding of the substrate fructose 6-phosphate (F6P) to PFK-1 PFK-1 exhibits a sigmoidal (S-shaped) v o versus [S] curve. This is due to the cooperativity of S binding. Increasing ADP concentrations lowers the apparent K m for F6P without affecting V max (activates PFK) The allosteric inhibitor PEP raises the apparent K m without changing V max

59 Prentice Hall c2002Chapter 559 Fig 5.19 Plots of initial velocity versus F6P for PFK-1 ADP is an allosteric activator of PFK-1 and lowers the apparent K m without affecting V max For a given F6P concentration the v o is larger in the presence of ADP

60 Prentice Hall c2002Chapter 560 Fig 5.20 Stereo views of PFK-1 from E. coli PFK-1 is a tetramer of identical chains. Products (yellow and green), allosteric activator ADP (red) (a) Single chain ribbon diagram (b) Tetramer: 2 blue, 2 purple chains

61 Prentice Hall c2002Chapter 561 B. General Properties of Allosteric Enzymes 1. Activities of allosteric regulator enzymes are changed by inhibitors and activators (modulators) 2. Allosteric modulators bind noncovalently to the enzymes that they regulate 3. Regulatory enzymes possess quaternary structure (continued next slide)

62 Prentice Hall c2002Chapter 562 General properties of regulatory enzymes (cont) 4. Regulatory enzymes have at least one substrate which has a sigmoidal v o versus [S] curve (positive cooperativity of multiple substrate binding sites) 5. There is a rapid transition between the active (R) and inactive (T) conformations 6. Substrates and activators may bind only to the R state while inhibitors may bind only to the T state

63 Prentice Hall c2002Chapter 563 Rapid transition exists between R and T states Addition of S increases concentration of the R state Addition of I increases concentration of the T state Activator molecules bind preferentially to R, leading to an increase in the R/T ratio

64 Prentice Hall c2002Chapter 564 Fig 5.21 Role of cooperativity of binding in regulation Addition of modulators alters enzyme activity Activators can lower K m, inhibitors can raise K m

65 Prentice Hall c2002Chapter 565 C. Two Theories of Allosteric Regulation Concerted theory - (symmetry-driven theory). Only 2 conformations exist: R and T ( symmetry is retained in the shift between R and T states) Subunits are either all R or all T R has high affinity for S, T has a low affinity for S Binding of S shifts the equilibrium toward all R state Binding of I shifts the equilibrium toward all T state

66 Prentice Hall c2002Chapter 566 Sequential Theory (ligand-induced theory) A ligand may induce a change in the structure of the subunit to which it binds Conformational change of one subunit may affect the conformation of neighboring subunits A mixture of both R (high S affinity) and T (low S affinity) subunits may exist (symmetry does not have to be conserved)

67 Prentice Hall c2002Chapter 567 Fig 5.22 Two models (a) Concerted model: subunits either all T state or all R state (b) Sequential model: Mixture of T subunits and R subunits is possible. Binding of S converts only that subunit from T to R

68 Prentice Hall c2002Chapter 568 Fig 5.23 Conformational changes during O 2 binding to hemoglobin Oxygen binding to Hb has aspects of both the sequential and concerted models

69 Prentice Hall c2002Chapter 569 D. Regulation by Covalent Modification Interconvertible enzymes are controlled by covalent modification Converter enzymes catalyze covalent modification Converter enzymes are usually controlled themselves by allosteric modulators

70 Prentice Hall c2002Chapter 570 Fig 5.24 General scheme for covalent modification of interconvertible enzymes

71 Prentice Hall c2002Chapter 571 Fig 5.25 Pyruvate dehydrogenase regulation Phosphorylation stabilizes the inactive state (red) Dephosphorlyation stabilizes the active state (green)

72 Prentice Hall c2002Chapter 572 5.11 Multienzyme Complexes and Multifunctional Enzymes Multienzyme complexes - different enzymes that catalyze sequential reactions in the same pathway are bound together Multifunctional enzymes - different activities may be found on a single, multifunctional polypeptide chain

73 Prentice Hall c2002Chapter 573 Metabolite channeling Metabolite channeling - “channeling” of reactants between active sites can occur when the product of one reaction is transferred directly to the next active site without entering the bulk solvent Can greatly increase rate of a sequence of reactions Can protect intermediates from solvent reactions Channeling is possible in multienzyme complexes and multifunctional enzymes

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