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Prentice Hall c2002Chapter 61 Principles of Biochemistry Fourth Edition Chapter 6 Mechanisms of Enzymes Copyright © 2006 Pearson Prentice Hall, Inc. Horton.

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Presentation on theme: "Prentice Hall c2002Chapter 61 Principles of Biochemistry Fourth Edition Chapter 6 Mechanisms of Enzymes Copyright © 2006 Pearson Prentice Hall, Inc. Horton."— Presentation transcript:

1 Prentice Hall c2002Chapter 61 Principles of Biochemistry Fourth Edition Chapter 6 Mechanisms of Enzymes Copyright © 2006 Pearson Prentice Hall, Inc. Horton Moran Scrimgeour Perry Rawn

2 Prentice Hall c2002Chapter 62 Chapter 6 Mechanisms of Enzymes Mechanisms - the molecular details of catalyzed reactions Enzyme mechanisms deduced from: Kinetic experiments Protein structural studies Studies of nonenzymatic model systems

3 Prentice Hall c2002Chapter The Terminology of Mechanistic Chemistry A reaction mechanism describes the details of a reaction Reactants, products and intermediates are identified Enzymatic mechanisms use the same symbolism used in organic chemistry

4 Prentice Hall c2002Chapter 64 Nucleophilic substitution reactions Nucleophilic species are electron rich, and electrophilic species are electron poor Types of nucleophilic substitution reactions include: (1) Formation of a tetrahedral intermediate by nucleophilic substitution (2) Direct displacement via a transition state

5 Prentice Hall c2002Chapter 65 Two types of nucleophilic substitution reactions Formation of a tetrahedral intermediate Direct displacement

6 Prentice Hall c2002Chapter 66 Cleavage reactions Carbanion formation Carbocation formation Free radical formation

7 Prentice Hall c2002Chapter 67 Oxidation-reduction reactions Electrons are transferred between two species Oxidizing agent gains electrons (is reduced) Reducing agent donates electrons (is oxidized) Formaldehyde is oxidized by oxygen (below)

8 Prentice Hall c2002Chapter Catalysts Stabilize Transition States Fig 6.1 Energy diagram for a single- step reaction

9 Prentice Hall c2002Chapter 69 Fig 6.2 Energy diagram for reaction with intermediate Intermediate occurs in the trough between the two transition states Rate determining step in the forward direction is formation of the first transition state

10 Prentice Hall c2002Chapter 610 Enzymes lower the activation energy of a reaction (1) Substrate binding Enzymes properly position substrates for reaction (makes the formation of the transition state more frequent and lowers the energy of activation) (2) Transition state binding Transition states are bound more tightly than substrates (this also lowers the activation energy)

11 Prentice Hall c2002Chapter 611 Fig 6.3 Enzymatic catalysis of the reaction A+B A-B

12 Prentice Hall c2002Chapter Chemical Modes of Enzymatic Catalysis A. Polar Amino Acid Residues in Active Sites Active-site cavity of an enzyme is lined with hydrophobic amino acids Polar, ionizable residues at the active site participate in the mechanism Anions and cations of certain amino acids are commonly involved in catalysis

13 Prentice Hall c2002Chapter 613 Table 6.1

14 Prentice Hall c2002Chapter 614 Table 6.2 pKa Values of amino acid ionizable groups in proteins GrouppK a Terminal  -carboxyl3-4 Side-chain carboxyl4-5 Imidazole6-7 Terminal  -amino7.5-9 Thiol8-9.5 Phenol  -Amino~10 Guanidine~12 Hydroxymethyl~16

15 Prentice Hall c2002Chapter 615

16 Prentice Hall c2002Chapter 616 B. Acid-Base Catalysis Reaction acceleration is achieved by catalytic transfer of a proton A general base (B:) can act as a proton acceptor to remove protons from OH, NH, CH or other XH This produces a stronger nucleophilic reactant (X: - )

17 Prentice Hall c2002Chapter 617 General base catalysis reactions (continued) A general base (B:) can remove a proton from water and thereby generate the equivalent of OH - in neutral solution

18 Prentice Hall c2002Chapter 618 Proton donors can also catalyze reactions A general acid (BH + ) can donate protons A covalent bond may break more easily if one of its atoms is protonated (below)

19 Prentice Hall c2002Chapter 619 C. Covalent Catalysis All or part of a substrate is bound covalently to the enzyme to form a reactive intermediate Group X can be transferred from A-X to B in two steps via the covalent ES complex X-E A-X + EX-E + A X-E + BB-X + E

20 Prentice Hall c2002Chapter 620 Sucrose phosphorylase exhibits covalent catalysis Step one: a glucosyl residue is transferred to enzyme *Sucrose + EnzGlucosyl-Enz + Fructose Step two: Glucose is donated to phosphate Glucosyl-Enz + P i Glucose 1-phosphate + Enz *(Sucrose is composed of a glucose and a fructose)

21 Prentice Hall c2002Chapter 621 D. pH Affects Enzymatic Rates Catalytic mechanisms are dependent upon the state of ionization of catalytic groups Plots of reaction velocity versus pH can indicate important ionizable catalytic groups

22 Prentice Hall c2002Chapter 622 Fig 6.4 pH-rate profile for papain The two inflection points approximate the pK a values of the two ionizable residues

23 Prentice Hall c2002Chapter 623 Fig 6.5 Papain’s activity depends upon ionizable residues: His-159 and Cys-25 (a) Ribbon model (b) Active site residues (N blue, S yellow)

24 Prentice Hall c2002Chapter 624 Fig. 6.6 Three ionic forms of papain. Only the upper tautomer of the middle pair is active

25 Prentice Hall c2002Chapter Diffusion-Controlled Reactions Enzyme rates can approach the physical limit of the rate of diffusion of two molecules in solution Under physiological conditions the encounter frequency is about 10 8 to 10 9 M -1 s -1 A few enzymes have rate-determining steps that are roughly as fast as the binding of substrates to the enzymes

26 Prentice Hall c2002Chapter 626 Table 6.4

27 Prentice Hall c2002Chapter 627 A. Triose Phosphate Isomerase (TPI) TPI catalyzes a rapid aldehyde-ketone interconversion

28 Prentice Hall c2002Chapter 628 Role of active site residues in TPI catalysis TPI has two ionizable active-site residues Glu acts as a general acid-base catalyst His shuttles protons between oxygens of an enzyme-bound intermediate

29 Prentice Hall c2002Chapter 629 Fig 6.7 Proposed mechanism for TPI General acid-base catalysis mechanism (4 slides)

30 Prentice Hall c2002Chapter 630 Fig 6.7 TPI mechanism (continued)

31 Prentice Hall c2002Chapter 631 Fig 6.7 TPI mechanism (continued)

32 Prentice Hall c2002Chapter 632 Fig 6.7 TPI mechanism (continued)

33 Prentice Hall c2002Chapter 633

34 Prentice Hall c2002Chapter 634 Fig 6.9 Energy diagram for the TPI reaction

35 Prentice Hall c2002Chapter 635 B. Superoxide Dismutase Superoxide dismutase is a very fast catalyst because: (1) The negatively charged substrate is attracted by a positively charged electric field near the active site (2) The reaction itself is very fast

36 Prentice Hall c2002Chapter 636 Two step reaction of superoxide dismutase An atom of copper bound to the enzyme is reduced and then oxidized

37 Prentice Hall c2002Chapter 637

38 Prentice Hall c2002Chapter Binding Modes of Enzymatic Catalysis Proper binding of reactants in enzyme active sites provides substrate specificity and catalytic power Two catalytic modes based on binding properties can each increase reaction rates over 10,000-fold : (1) Proximity effect - collecting and positioning substrate molecules in the active site (2) Transition-state (TS) stabilization - transition states bind more tightly than substrates

39 Prentice Hall c2002Chapter 639 Binding forces utilized for catalysis 1. Charge-charge interactions 2. Hydrogen bonds 3. Hydrophobic interactions 4. Van der Waals forces

40 Prentice Hall c2002Chapter 640 A. The Proximity Effect Correct positioning of two reacting groups (in model reactions or at enzyme active sites): (1) Reduces their degrees of freedom (2) Results in a large loss of entropy (3) The relative enhanced concentration of substrates (“effective molarity”) predicts the rate acceleration expected due to this effect

41 Prentice Hall c2002Chapter 641 Effective molarity k 1 (s -1 ) Effective molarity = k 2 (M -1 s -1 )

42 Prentice Hall c2002Chapter 642 Fig 6.11 Reactions of carboxylates with phenyl esters Increased rates are seen when the reactants are held more rigidly in proximity (continued next slide)

43 Prentice Hall c2002Chapter 643 Fig (continued)

44 Prentice Hall c2002Chapter 644 B. Weak Binding of Substrates to Enzymes Energy is required to reach the transition state from the ES complex Excessive ES stabilization would create a “thermodynamic pit” and mean little or no catalysis Most K m values (substrate dissociation constants) indicate weak binding to enzymes

45 Prentice Hall c2002Chapter 645 Fig 6.12 Energy of substrate binding If an enzyme binds the substrate too tightly (dashed profile), the activation barrier (2) could be similar to that of the uncatalyzed reaction (1)

46 Prentice Hall c2002Chapter 646 C. Induced Fit Induced fit activates an enzyme by substrate- initiated conformation effect Induced fit is a substrate specificity effect, not a catalytic mode Hexokinase mechanism requires sugar-induced closure of the active site Glucose + ATP Glucose 6-phosphate + ADP

47 Prentice Hall c2002Chapter 647 Fig 6.13 Stereo views of yeast hexokinase Yeast hexokinase contains 2 domains connected by a hinge region. Domains close on glucose binding. (a) Open conformation (b) Closed conformation

48 Prentice Hall c2002Chapter 648 D. Transition-State (TS) Stabilization An increased interaction of the enzyme and substrate occurs in the transition-state (ES ‡ ) The enzyme distorts the substrate, forcing it toward the transition state An enzyme must be complementary to the transition-state in shape and chemical character Enzymes may bind their transition states to times more tightly than their substrates

49 Prentice Hall c2002Chapter 649 Transition-state (TS) analogs Transition-state analogs are stable compounds whose structures resemble unstable transition states Fig Phosphoglycolate, a TS analog for the enzyme triose phosphate isomerase

50 Prentice Hall c2002Chapter 650 Fig 6.15 Inhibition of adenosine deaminase by a TS analog

51 Prentice Hall c2002Chapter 651 Catalytic antibodies Fig 6.16 Antibody- catalyzed amide hydrolysis ( a) TS analog antigen (b) Reaction catalyzed by the catalytic antibody

52 Prentice Hall c2002Chapter Lysozyme Binds an Ionic Intermediate Tightly Lysozyme binds polysaccharide substrates (the sugar in subsite D of lysozyme is distorted into a half-chair conformation) Binding energy from the sugars in the other subsites provides the energy necessary to distort sugar D Lysozyme binds the distorted transition-state type structure strongly

53 Prentice Hall c2002Chapter 653 Fig 6.17 Bacterial cell-wall polysaccharide Lysozyme cleaves bacterial cell wall polysaccharides (a four residue portion of a bacterial cell wall with lysozyme cleavage point is shown below)

54 Prentice Hall c2002Chapter 654 Fig 6.18 Lysozyme from chicken with a triaccharide molecule (pink)

55 Prentice Hall c2002Chapter 655 Fig 6.19 Conformations of N-acetylmuramic acid (a) Chair conformation (b) D-Site sugar residue is distorted into a higher energy half- chair conformation

56 Prentice Hall c2002Chapter 656 Lysozyme reaction mechanisms 1. Proximity effects 2. Acid-base catalysis 3. TS stabilization (or substrate distortion toward the transition state)

57 Prentice Hall c2002Chapter 657 Fig 6.20 Mechanism of lysozyme (2 slides)

58 Prentice Hall c2002Chapter 658

59 Prentice Hall c2002Chapter Properties of Serine Proteases A. Zymogens Are Inactive Enzyme Precursors Digestive serine proteases including trypsin, chymotrypsin, and elastase are synthesized and stored in the pancreas as zymogens Storage of hydrolytic enzymes as zymogens prevents damage to cell proteins Zymogens are activated by selective proteolysis

60 Prentice Hall c2002Chapter 660 Fig 6.21 Activation of some pancreatic zymogens

61 Prentice Hall c2002Chapter 661 Fig 6.22 Stereo view of the backbones of chymotrypsinogen and  -chymotrypsin Chymotrypsinogen (blue),  -chymotrypsin (green) Catalytic Asp, His, Ser (red), Ile-16, Asp-194 (yellow)

62 Prentice Hall c2002Chapter 662 B. Substrate Specificity of Serine Proteases Many digestive proteases share similarities in 1 o,2 o and 3 o structure Chymotrypsin, trypsin and elastase have a similar backbone structure Active site substrate specificities differ due to relatively small differences in specificity pockets

63 Prentice Hall c2002Chapter 663 Fig 6.23 Comparison of the backbones of (a) chymotrypsin (b) trypsin and (c) elastase Backbone conformations and active-site residues (red) are similar in these three enzymes

64 Prentice Hall c2002Chapter 664 Fig 6.24 Binding sites of chymotrypsin, trypsin, and elastase Substrate specificities are due to relatively small structural differences in active-site binding cavities

65 Prentice Hall c2002Chapter 665 C. Serine Proteases Use Both the Chemical and Binding Modes of Catalysis  - Chymotrypsin active site groups include: Ser identified by covalent DFP labeling His-57 - identified by affinity labeling with TosPheCH 2 Cl Asp identified by X-ray crystallography

66 Prentice Hall c2002Chapter 666 Fig 6.25 Stereo view of the catalytic site of chymotrypsin Active-site Asp- 102, His-57, Ser- 195 are arrayed in a hydrogen- bonded network (O red, N blue)

67 Prentice Hall c2002Chapter 667 Fig 6.26 Catalytic triad of chymotrypsin Imidazole ring (His-57) removes H from Ser-195 hydroxyl to make it a strong nucleophile (-CH 2 O - ) Buried carboxylate (Asp-102) stabilizes the positively- charged His-57 to facilitate serine ionization

68 Prentice Hall c2002Chapter 668 Fig 6.27  -Chymotrypsin mechanism (8 slides) Step (1): E + S

69 Prentice Hall c2002Chapter 669 Fig 6.27 (E-S)

70 Prentice Hall c2002Chapter 670 Fig 6.27 (E-TI 1 )

71 Prentice Hall c2002Chapter 671 Fig 6.27 (Acyl E + P 1 )

72 Prentice Hall c2002Chapter 672 Fig 6.27 (Acyl E + H 2 O) (4)

73 Prentice Hall c2002Chapter 673 Fig 6.27 (E-TI 2 ) (5)

74 Prentice Hall c2002Chapter 674 Fig 6.27 (E-P 2 ) (6)

75 Prentice Hall c2002Chapter 675 Fig 6.27 (E + P 2 )

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