BMB Lecture Covalent Catalysis 2. General Acid-base catalysis

Transcription

1 BMB Lecture 3 1. Covalent Catalysis 2. General Acid-base catalysis

2 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.

3 Evidences for A Covalent Intermediate Indirect Evidences: Different substrates with diverse chemical reactivities react at the same rate on the enzyme 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**

4 Why Is It Advantageous to Use Covalent Catalysis?

5 Chemical logic to use covalent catalysis G E A-B E-B +A E B-X Reaction Coordinate 1. The catalyst has a better nucleophile than the substrate group (X). 2. The intermediate is more reactive than the substrate. 3. The intermediate is less stable than the product.

6 Nucleophilic Catalysis by Imidazole 1. 3 amines are more reactive than H - 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.

7 Electrophilic Catalysis: Schiff base formation Activated nucleophile Electron sink

8 Example 1. Acetoacetate decarboxylase - H 3 C C H2 - H 3 C CH 2 + C E NH 2 CH 2 CH 3 H + E N H + CH 2 CH3 C 2 E N H CH 2 CH 3 H + E N H + CH 3 H 2 E NH 2 + H + + CH 3 CH 3 CH 3

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

10 Chemistry with pyridoxal schiff base Highly reactive intermediate 1. Racemization 2. Decarboxylation H H,1'0 R I c r\ "cj "o- I 6- + N H

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

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

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

14 Nucleophile Positioning and Thio-effects in NDP Kinase

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

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

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

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

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

20 How good are the designed enzymes?

21 What s wrong with RA 95.0? rosetta design crystal structure a L6 L1 L7 H 2 Glu53 Lys210 - 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

22 General Acid Base Catalysis

23 Catalysis of Acetylimidazole Hydrolysis by Imidazole Buffer H 3 C N H N H 3 C H + NH N H

24 Catalysis of Acetylimadazole Hydrolysis by Imidazole Buffer H 3 C H N H N N NH H 3 C δ- H δ- N H + N N NH H + H 3 C + HN H + NH N NH Importance of General Acid-Base Catalysis Facilitates proton transfer to avoid formation of highly unstable intermediates. Does so without the formation of H - or H + ions.

25 Proton Transfer from Carbon Acids Ketone Enolization

26 Proton Transfer from Carbon Acids Highly unfavorable deprotonation renders the activation barrier very high

27 Proton Transfer from Carbon Acids Barrier for proton transfer is much larger for C-H than for acidic protons

28 Proton Transfer from Heteroatoms pk a ~ 14 ΔG = 9.5 kcal/mol

29 Proton transfer from Heteroatoms pk a ~ 14 ΔG = 9.5 kcal/mol pk a ~ 2 ΔG = 12.6 kcal/mol

30 Proton transfer from Heteroatoms pk a ~ 14 ΔG = 9.5 kcal/mol pk a ~ 2 ΔG = 12.6 kcal/mol GABC avoids the formation of highly unstable intermediates

31 Principle of Microscopic Reversibility: Both the forward and reverse reactions must go through the same transition state H H H C N H A H δ+ δ- C N H A H + H C NH A - fast H H + C N H A - If a catalyst donates a proton in the forward reaction, its conjugate base act to remove the same proton in the reverse reaction

32 Measuring the Efficiency of GABC: Brønsted Relationship log k B = log k 0 + β(pk a ) Brønsted slope measures degree of proton transfer log k HA = log k 0 - α(pk a )

33 Efficiency vs Effectiveness of GABC Most effective general acid/base has pk a ~ 7 under physiological conditions

34

35 Interpreting ph-rate Profiles 1 semi-log plot 1 log plot k obsd 0.4 k obsd ph ph A proton needs to be lost (or gained) in going from the reaction ground state to More specific interpretations regarding pk a and site of general acid / base catalysis are subject to simplifying assumptions.

36 Assumptions in ph-rate Analysis 1. nly one state of enzyme is active (no parallel or diversionary pathways) 2. All protonation equilibria are fast compared to enzymatic reaction steps (i.e., no sticky protons) 3. No change in rate-limiting step over the ph range (kinetic vs real pk a ) 4. Enzyme is not inactivated by changes in ph Preferred: - ph dependence of an elementary step - Direct titration of enzymatic group

37 ph-rate Profiles in Enzymatic Catalysis 1 semi-log plot 1 log plot k obsd 0.4 k obsd ph pk a ph pk a H + EH K A E + S k E products k obsd = k E [E] E total = k E 1 1+[H + ]/K A

38 ph-rate Profiles in Enzymatic Catalysis 1 two pk A 's 0.1 k obsd pk A 1 pk A ph H + H + EH 2 K A 1 EH + S K A 2 k E products k obsd = k EH [EH] E total = k EH K A 1 [H + ] K A 1 KA 2 + KA 1 [H + ]+[H + ] 2 E

39 Case 1. Chymotrypsin Hydrolysis of anilide (slow substrate) Renard & Fersht, Biochemistry 12: 4713 (1973)

40 Case 1. Chymotrypsin E + S ES E-I E + P 2 P 1 K S k 23 k 34 k 23 k 34 pk a = 6.8 for both steps

41 Case 1. Chymotrypsin bserved pk a ~ 6.8 Identity of general base? Imidazole (active site His57) most likely but can also be other groups with perturbed pk a Evidences for His57 being the general base Alkylation of active site imidazole by substrate analog inactivates chymotrypsin and abolishes ph dependence. ph dependence of alkylation shows the same pk a.

42 Case 1. Chymotrypsin Direct titration of active site His57 by NMR

43 Case 1. Chymotrypsin Model for general base catalysis by His57 X R H Im X δ- δ+ H Im X R + H Im Ser R Ser Ser R X H Im δ- δ+ X H Im H R X Im Ser Ser R Ser

44 Criteria for General Acid/Base Catalyst Mutation of the candidate residue abolishes ph dependence of the reaction pk A of the candidate residue is the same as the pk A observed in ph-rate profiles Reaction of substrates with better nucleophile or leaving group is less dependent on the general acid/base

45 Case 2. Tetrahymena Group I Ribozyme Self-splicing Ribozyme-catalyzed endonuclease reaction

46 Case 2. Tetrahymena Group I Ribozyme E S + G > products Kinetic or real pk a?

47 Case 2. Tetrahymena Group I Ribozyme Kinetic or real pk a? Test with slower substrates

48 Case 2. Tetrahymena Group I Ribozyme Test with slower substrates (-1d, rs o and -1F, rsr)

49 Case 2. Tetrahymena Group I Ribozyme E S + G > products A proton must be lost in going from the ground state to the transition state (most likely, 3 -H of G) Chemical step is rate-limiting below ph 7 Above ph 7, a different step is rate-limiting (G binding)

50 Recommended Readings CRC Critical Reviews in Biochemistry (1976): 165