BMB Lecture 2

Transcription

1 BMB Lecture 2 How to map transition state Covalent Catalysis

2 How to Map Transition States 1. Linear Free Energy Relationship 2. Kinetic Isotope Effects 3. Transition state analogues

3 Linear Free Energy Relationship Correlate reaction rate with other equilibrium processes DG = -RT lnk DG = -RT lnk = -RT ln k To the extent that such a correlation holds, it may be inferred that the same factors contribute to both processes. Brønsted equation: log k = b pk a + C Brønsted slope Measures the amount of bond formation/breakage in

4 Example: Phosphoryl Transfer b nuc => Little bond formation with incoming nucleophile Herschlag & Jencks, JACS 1989 Factors other than pk a dictate nucleophilic reactivity - polarizability (softness) - oxidation potential - size - electronegativity

5 Example: Phosphoryl Transfer b LG b LG 1.2 Extensive bond breaking with leaving group

6 Reaction Coordinate Diagram for Phosphoryl Transfer (Associative, Pentavalent transition state) (Dissociative, metaphosphate-like transition state)

7 Kinetic Isotope Effects Infrared stretching frequencies: n = 2900 cm -1 (4.15 kcal/mol) for C-H bonds n = 2100 cm -1 (3.0 kcal/mol) for C-D bonds } => k H /k D 7

8 Types of Isotope Effects Solvent vs Substrate Isotope Effects exchangeable protons (O-H, N-H, S-H) non-exchangeable protons (C-H) Heavy Atom Isotope effects 13 C/ 12 C = 1.06; 18 O/ 16 O = 1.06; 15 N/ 14 N = 1.04 Primary vs Secondary Isotope Effects extent of bond breaking bond order/re-hybridization If (1) the bond is completely broken in ; (2) occurs during the rate-limiting step

9 Example 1. Hydrolysis of Aryl Phosphates Spontaneous hydrolysis: k ( 16 O) / k ( 18 O) = Substantial P-O bond breaking to b-g bridge oxygen in the transition state

10 Example 2. Protein Tyrosine Phosphatase 18 O nonbridge measures bond order metaphosphate-like small inverse IE pentavalent (associative) normal IE 18 O bridge measures extent of P-O bond cleavage 15 N measures charge developed on leaving group

11 KIE for hydrolysis of pnpp in solution 2% KIE on 18 O bridge - substantial P-O bond cleavage in Hengge et al, JACS 116, 5045 (1994)

12 KIE for hydrolysis of pnpp in solution 2% KIE on 18 O bridge - substantial P-O bond cleavage in Small, inverse KIE on 18 O nonbridge - metaphosphate-like Hengge et al, JACS 116, 5045 (1994)

13 KIE for hydrolysis of pnpp in solution 2% KIE on 18 O bridge - substantial P-O bond cleavage in Small, inverse KIE on 18 O nonbridge - metaphosphate-like KIE on 15 N substantial charge accumulation on O bridge Hengge et al, JACS 116, 5045 (1994)

14 KIE for hydrolysis of pnpp in solution 2% KIE on 18 O bridge - substantial P-O bond cleavage in Small, inverse KIE on 18 O nonbridge - metaphosphate-like KIE on 15 N substantial charge accumulation on O bridge 15 N and 18 O bridge KIE decreases upon protonation of g- phosphate proton migration from O nonbridge to O bridge (acid catalysis renders more associative) Hengge et al, JACS 116, 5045 (1994)

15 KIE on Protein Tyrosine Phosphatase 1.8% KIE on 18 O bridge substantial P-O bond cleavage in Small, inverse KIE on 18 O nonbridge metaphosphate-like No 15 N KIE negative charge on leaving group is neutralized 15 N and 18 O bridge KIE increases upon mutation of D181, suggesting that D181 acts as a general acid to protonate leaving group Hengge et al, Biochemistry 34, (1995)

16 ATP / GTP hydrolysis Admiraal & Herschlag, Chem. Biol. 2: 729 (1995)

17 Evaluation of Catalytic Models 1. General Base Catalysis? For reaction involving a dissociative, reaction has little dependence on basicity of the nucleophile Proton transfer from HOH (pk a 14) to Gln61 (pk a -2) is very unfavorable Role of Gln61? - positioning - facilitate proton transfer after

18 Evaluation of Catalytic Models 2. Phosphoryl Group Acts as A General Base? Proton donation to phosphoryl oxygen will destabilize a dissociative

19 Evaluation of Catalytic Models 3. Stabilization of charge on g-phosphoryl? Negative charge does not accumulate on g- phosphoryl oxygen in a dissociative

20 Evaluation of Catalytic Models 4. Stabilization of charge on b-phosphoryl? Can provide a small rate enhancement

21 Evaluation of Catalytic Models 4. Stabilization of charge on b-g bridge oxygen? Can potentially provide a large rate enhancement

22 Conserved Active Site Interactions with Bridge Oxygen GMPPNP GDP

23 F value Analysis to Study Protein Folding DDG U = DG U DG U = DG F - DG solv DDG U = DG U - DG U = DG F - DG F = DDG U /DDG U = (DG F DG )/(DG F DG solv ) Matouschek et al, Nature 340: 122 (1989)

24 F value Analysis to study Protein Folding F = DDG U /DDG U = (DG F DG )/(DG F DG solv ) 1. Target site is exposed to solvent in the to the same extent as in the unfolded state DG = DG solv => F = 1 2. Interaction energies in are similar to those in the folded state DG = DG F => F = 0 3. DG solv ~ 0 with small changes in hydrophobic side chains F = (DG F - DG ) / DG F

25 F value Analysis of Barnase folding

26 F value Analysis of Barnase folding

27 F value analysis of Barnase folding Structure of : hydrophobic core and secondary structure formed, but the N-cap and loops largely unfolded.

28 1. Best nucleophiles in biology: - Amines (RNH 2 ), alkoxides (RO - ), thiolates (RS - ), carbanions ( R 3 C - ) - Weaker but sometimes: carboxylates (RCO 2- ), phosphate (HPO 4 3- ) 2. Best electrophiles in Biology: - Phosphorus esters (HPO 3 -OR), - Carbonyls (RC=ONHR, RCO 2 R, etc.) 3. Good leaving groups in Biology: - Phosphates ( HPO 4 3- ), - Protonated amines ( NH 3+ ) - Protonated alcohols ( OH 2+ )

29

30 Strategies for Enzymatic Catalysis Covalent Catalysis General Acid-base Catalysis Metal Ions Electrostatics Entropy Putting it all together the intrinsic bind energy

31 Covalent Catalysis in the History of Enzymology enzymes are not chemical individuals, but that various kinds of bodies may have conferred upon them properties which cause them to behave like enzymes; so that we have to deal with properties rather than substances. The action can even be exerted at a distance. Balyliss, The Nature of Enzyme Action, 2nd ed. The assumption of this new force is detrimental to the progress of science, since it appears to satisfy the human spirit, and thus provides a limit to further research. Liebig, on Berzelius vitalistic theory of enzymes

32 Covalent Catalysis in the History of Enzymology The discovery that enzymes react chemically with their substrates to form covalent intermediates has done more than anything else to dispel mysterious mechanisms and vitalistic theories of enzyme action because it suggest that the mechanism of enzyme action is not fundamentally different from that of any other chemical reactions. The rapid progress in describing some enzymatic reactions in chemical terms and in the application of physical organic chemistry techniques to enzymoology has led to a swing of the pendulum of opinion W. P. Jencks, Catalysis in Chemistry and Enzymology, 2nd ed.

33 Covalent Catalysis 1. Demonstration of a covalent intermediate 2. Chemical and Energetic rationale

34 Initial Observation: An Initial Burst of Product Formation Stoichiometric with [E] Models: (1) Rate-limiting product release (2) Product Inhibition (3) Substrate-induced inactivation of enzyme (4) Two-step reaction with ratelimiting breakdown of Ac-E Hartley & Kilby, 1954

35 k cat is identical for substrates with fold difference in chemical reactivity Kirsch & Igelstrom, 1966

36 Direct Observation of Acyl-E at low ph ph 2.42, [E] > K S; [S] ~ 5[E] A. N-acetyl-L-tryp (P) B. N-acetyl-L-tryp-methyl ester Formation of Acyl-E k 2 = k cat deacylation of Acyl-E Kezdy et al, Biochemistry 86: 3690 (1964)

37 Crystal Structures of Acy-E Indoleacryloyl-a-chymotrypsin, ph 4.0 His57 Ser195 Henderson, J. Mol. Biol. 54: 341 (1970)

38 So Acyl-E is Demonstrated at low ph What about physiological reactions?

39 Millisecond kinetic measurement of the Chymotrypsin reaction Four kinetic phases: 1. Substrate binding to E: too fast to observe 2. Rapid formation of Acyl-E intermediate (< 0.5 sec) 3. Steady-state (0.5 5 sec) 4. Decomposition of Acyl-E (> 5 sec) McConn et al, JBC 246: 2918 (1971)

40 What if Acyl-E do Not Accumulate? Ester substrates: deacylation is rate-limiting Amide substrates: formation of acyl-e is rate-limiting Product Ratios from a Common Intermediate If the product ratios are the same for both amide and ester substrates, then reaction of both substrates involve a common intermediate

41 Product Partitioning in Chymotrypsin substrate Fastrez & Fersht, Biochemistry 12: 2025 (1973)

42 Evidences for A Covalent Intermediate Direct Evidences: Direct observation of formation and disappearance of an intermediate Kinetic competence: Rate constants for formation and disappearance of the intermediate must be at least as fast as the rate constant for the overall reaction Isolation and characterization of the intermediate The intermediate can be chased to the product with kinetic competence.

43 Evidences for A Covalent Intermediate Indirect Evidences: Different substrates with various reactivities form products at the same rate If different substrates share a common intermediate, product partitioning from E-I is the same for both substrates Stereochemistry: - Retention: 2-steps - Inversion: 1-step Parallel (ping-pong) kinetics

44 Why Is It Advantageous to Use Covalent Catalysis?

45 Chemical Logic to Use Covalent Catalysis 1. The catalyst has a higher nucleophilic reactivity than the final acceptor (X). 2. The Intermediate is more reactive than the substrate. 3. The Intermediate is less stable than the product.

46 Nucleophilic Catalysis by Imidazole 1. 3 amines are more reactive than OH - in nucleophilic attacks. 2. pk a ~ 7 the strongest base that can exist at neutral ph. 3. Acetylimidazole is highly susceptible to nuclephilic attacks. 4. Acetylimidazole is thermodynamically less stable compared to amines and esters.

47 Electrophilic Catalysis: Schiff base formation Activated nucleophile Electron sink

48 Example 1. Acetoacetate decarboxylase

49 Electrophilic Catalysis by Pyridoxal phosphate condense with Amino acid Pyridoxal phosphate Schiff base Each group around Ca can be cleaved; The anion is stabilized by the pyridine ring (electron sink)

50 Chemistry with pyridoxal schiff base Highly reactive intermediate 1. Racemization 2. Decarboxylation

51 Chemistry with pyridoxal schiff base 3. Interconversion of side chains RX = SH, OH, indole Allow interconversion of ser, thr, cys, trp, cystathione, etc.

52 but many enzymes don t use reactive nucleophiles or cofactors in covalent catalysis Positioning of the Nucleophile is Important

53 Nucleophilic Activation by Positioning in NDP Kinase Admiraal et al, Biochemistry 38: 4701 (1999)

54 Nucleophile Positioning and Thio-effects in NDP Kinase

55 Computationally designed Retroaldolase Lin et al, Science 319:1387 (2008)

56 Design motif 1 12 designs 2 with schiff base formation None reactive

57 Design motif 2 10 designs 1 with schiff base formation None reactive

58 Design motif 3 14 designs 10 with schiff base formation 8 had retroaldolase activity

59 Design motif 4 36 designs 20 with schiff base formation 23 had retroaldolase activity

60 How good are the designed enzymes?

61 What s wrong with RA 95.0? rosetta design crystal structure - Naphthal ring is rotated Largest deviation observed with Rosetta-designed loops (L1, L6, L7) that bracket the active site. - Substrate points away from water and Glu153