Enzymes! Accelerate reactions by x 10 6 (and up to x ) Specific with respect to reaction catalized.

Transcription

1 Enzymes! Accelerate reactions by x 10 6 (and up to x ) Specific with respect to reaction catalized. Selective with respect to reagent recognized. Cartoon Guide to Genetics Gonick & Wheelis 1

2 Microreview Categories of catalytic mechanisms Entropy loss in ES formation, Destabilization of ES via strain, desolvation, Estat Covalent catalysis General Acid/Base Metal ion catalysis Proximity and orientation (entropic) 2

3 Low-barrier -bonds (not a mechanism of catalysis) Fig shifts in pks, + transfer. Bond (e - ) redistribution

4 Metal ions, (not a mechanism) But crucial for enforcing proximity, Stabilizing charge (electrostatic ) Activating a nucleophile REDX roles (not covered) Fig

5 Example of a metallo-enzyme (non-redox) C + C + is- Zn 2+ - is- - Zn 2+ is- is- -is -is Carbonic anhydrase

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8 Example 1 Serine proteases Trypsin Fig 5.16 Acetyl choline esterase. Structure of an inhibited complex.

9 Catalytic triad Ser is57 - Asp102Distant in the primary structure brought together by tertiary structure ALS: separate Asp189 - substrate binding and specificity TEME: binding via side chains of substrate. Separate from the site of reaction. Different residues responsible for k cat and K M. Minimizes the extent to which substrate binding interpheres with transition state stabilization. X ptimal S-b Separate X -b. EX S ES ES

10 Burst kinetics: regeneration of the enzyme is slower than release of 1st product Fig

11 Acetate release is the rate-limiting step. Fig

12 Trapping the intermediate (vs. suicide inhibitors) If hydrolysis of E- X is T slow, the life-time of the intermediate can get so long that the enzyme is essentially stopped in intermediate form (eg. DIFP). Fig ote: DIFP attacks LY the AS ser, not many the others in the enzyme!! (another clue that Ser195 is special).

13 The catalytic triad act together to produce a better nucleophile Ser is a strong nucleophile (pk 13. Table 4.1) but is masked by protonation. Fig is effectively deprotonates Ser, But is is a weaker base than Ser!! -bond to Asp increases the basicity of is. Asp s special role is T to position is (Asn does too)**

14 Three nucleophiles augment one-another -

15 Impossible: is pk is too low. ΔG deprot = -RT ln K deprot. = 2.3RT pk RT(6.0) + 2.3RT(13) = 5.8 kj/mol * (13-6) = + 40 kj/mol BUT, is has another pk, 14

16 But, is has another pk,

17 Concerted proton transfer (avoid thinking of + as billiard ball: + can be shared) Effective pk? - - Strong -bd - -

18 Concerted proton transfer (avoid thinking of + as billiard ball: + can be shared) Effective pk? - - Loose weak -bd Strong -bd Gain strong -bd - Inverse of pk=6 -

19 Concerted proton transfer (avoid thinking of + as billiard ball: + can be shared) Effective pk? - - Loose weak -bd Strong -bd Gain strong -bd - - ΔG = +10 kj/mol - 5.8*6kJ/mol - 50 kj/mol = kj/mol kj/mol = -2.3RT pk eff pk eff =12.8

20 Concerted proton transfer (avoid thinking of + as billiard ball: + can be shared) Effective pk? - - Loose weak -bd Strong -bd Gain strong -bd - - Chicken / egg: is + has a pk 6, near Asp (4.5) is has a pk near 14, not near Asp. nly upon protonation of is does the -bond get strong

21 Chymotrypsin Fig 14.25

22 Chymotrypsin xyanion hole Fig 14.25

23 Tetrahedral intermediate with anionic. Fig. 1. Stick diagrams overlaid with 2Fo-Fc density maps contoured at 1 {sigma} (gray mesh), showing covalent attachment of substrate ligands to trypsin xianion hole stabilizes tetrahedral transition state ELECTRSTATICS Radisky, Evette S. et al. (2006) Proc. atl. Acad. Sci. USA 103,

24 Fig. 2. Structural views and superpositions focusing on enzyme and substrate residues and water molecules involved in reaction Radisky, Evette S. et al. (2006) Proc. atl. Acad. Sci. USA 103,

25 Example II Aspartate proteases Fig p dependence: general acid and general base contributions. X- ray structure: A and B: are Asp and Asp -. BUT Asp is not a strong base, cannot deprotonate water.

26 Low-Barrier -bond model Fig Coupling between two Asp: first pk is raised. This holds a proton which can stabilize -. A proton wire, or fluid. + tunneling.

27 transition state analogs in the treatment of AIDS

28 Example III: Lysozyme Fig

29 Regulation: a distinctive features of enzymes Factors that determine the actual rate of a catalized reaction: Availability of S and cofactors. Accumulation of P (significance of the reverse reaction). Regulation of the amount of enzyme present, via control over rate of enzyme production and degradation. Covalent modification of the enzyme that alters the KM and/or kcat. (phosphorylation, cleavage). Mix-and-match of subunits with different inherent KM and/or kcat. Allosteric regulation of enzyme activity, altering KM and/or kcat: Allosteric : occurs at another site, effector may have other (different) shape. 29

30 Coupled binding of A and B KA KB A + P + B A + PB KAB AP + B APB K A B Dissociation constants, K, and associated free energies of dissociation. K B A -ΔGA -ΔG A B = -ΔGB -ΔG B A -ΔGA = ΔG A B -ΔGB -ΔG B A A!G d,b B "!G d,b =!G d,a "!G d,a = # Coupling

31 Coupled binding of A and B A + P + B KA AP + B KB KAB K A B A + PB APB K B A -ΔGAB = -ΔGA -ΔG A B and -ΔG A B = -ΔGB -ΔG B A +ΔGA = -ΔGB - δ -ΔGAB = -ΔGA -ΔGB - δ! = "G d,ab # "G d,a # "G d,b This is the free energy associated with APB + P AP + PB +ve δ favors combined (cooperative) binding.

32 Fractional Saturation A + P + B KA AP + B KB A + PB APB K A B K A K B A = K B K A B K B A [PB] + [APB] [P] Tot = [B] K B! " # 1 + [A] K A + [B] K B 1+ [A] K A B! " # $ % & 1+ [A] K A B $ % & 32 K

33 eterotropic cooperativity 1.2 fraction of P with B bound Fraction of sites with B bound PB+APB/Ptot A=0 A=.25 mm A=.5 mm A=2 mm A=10 mm KB=1 mm K A B=0.1 mm KA=5 mm K B A=0.5 mm [B] [B] (mm) ( in mm)

34 alf-saturation [PB] + [APB] [P] Tot = 1 2 == [B] 1/2 K B! " # 1+ [A] K A + [B] 1/2 K B 1 + [A] K A B! " # $ % & 1+ [A] K A B $ % & 1 + [A] K A + [B] 1/2 K B! " # 1+ [A] K A B $ % & = 2[B] 1/2 K B! " # 1 + [A] K A B $ % & K B! " # 1 + [A] K A = [B] 1/2 K B 1+ [A] K A! " # $ % & = [B]! 1/2 " # 1 + [A] K A B 1 + [A] K A B $ % & $ % &! 1 + " # [B] 1/2 = K B! " # [A] K A 1 + [A] K A B $ % & $ % &

35 Cooperativity! 1+ " # [B] 1/2 = K B! " # [A] K A 1+ [A] K A B What does this become when K B A=KA (no coupling)? K B A<KA (effector A binding is cooperative with ligand B binding) $ % & $ % & A!G d,b B "!G d,b =!G d,a "!G d,a = # Coupling KA/K B A = e δ always positive, positive δ KA > K B A

36 Cooperative binding of a single ligand type (B)! [P] tot = [P] 1+ [B] + [B]! 1 + 2[B] $ $ # B K B " # K B % & & " % K B [PB] + [BP] + 2[BPB] [P] Tot = [B] K B! " # 1+ [B] K B 2 + 2[B] K B B! " # $ % & 2 + 2[B] $ B K B % &

37 omotropic cooperativity fraction sites filled Cooperative system saturates in a very narrow concentration range. second 10x tighter same tightness 100 x tighter [B]

38 1.2 1 fraction sites filled second 10x tighter same tightness 100 x tighter 0.2 [B]

39 1.2 1 fraction sites filled Sigmoidal signature of homotropic cooperativity. second 10x tighter same tightness 100 x tighter 0.2 [B]