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The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 9 Isomerizations Isomerizations Conversion of one molecule into another with the same formula • Hydrogen shifts to the same carbon: [1,1]-H shift • Hydrogen shifts to the adjacent carbon: [1,2]-H shift • Hydrogen shifts to two carbon atoms away: [1,3]-H shift [1,1]-Hydrogen Shift Racemase with no cofactors Glutamate racemase Not PLP - no visible absorbance Not pyruvoyl - acid hydrolysis gave no pyruvate No M2+ - EDTA has no effect No acyl intermediates - no 18O wash out of [C18O2H]Glu Not oxidation/reduction - 2H is incorporated into C-2 from 2H O 2 Therefore deprotonation/reprotonation mechanism [1,1]-Hydrogen Shift Amino acid racemases (A) One-base mechanism for racemization (epimerization), (B) Two-base mechanism for racemization (epimerization) R R NH3 H + NH3+ -OOC COO- A -OOC H NH3 + -OOC COO- a H B B R R H Scheme 9.1 H B B B R +H N 3 BH a B R +H N 3 NH3+ b b H B -OOC BH H B One base: substrate proton transferred to product Two base: incorporated proton from solvent With Glu racemase: solvent deuterium in product, not substrate (B) also, primary kinetic isotope effect with [2-2H]Glu An “Overshoot” Experiment with (R)-(-)-glutamate to Test for a Two-base Mechanism for Glutamate Racemase in D2O 100 60 20 0 E ll ip ti ci ty (m il li d eg re e) -20 -60 -100 1000 Figure 9.1 2000 Time (sec) 3000 Another Test for a Two-Base Mechanism Elimination of HCl from threo-3-chloroglutamic acid by the C73A and C184A mutants for glutamate racemase COO- H Cl Cl R R S S S H NH3+ NH3+ C184 COO- H COO- C73A COO- COO- H H Cl R R -OOC NH3+ NH3+ COO- +H N 3 D2O D COO- D2O COO- D H Scheme 9.2 COO- H S S H S C73 9.1 C184A Cl -OOC COO- H COO- H H -OOC O 9.2 COO- O 9.2 -OOC H NH3+ Proposed Mechanism for Proline Racemase S S H H H N HO HO H H N S H SH O S H N HO H O S H H N HOOC O ‡ S H S 9.3 Scheme 9.3 Inactivation by ICH2COO- only after a reducing agent (RSH or NaBH4) is added Reduces active site disulfide to dithiol Transition State Analogue Inhibitor Because substrates bind tightest at the transition state of the reaction, a compound resembling the TS‡ structure would be more tightly bound -OOC H N 9.4 resembles 9.3 TS‡ analogue inhibitor for Pro racemase Pyridoxal 5-Phosphate (PLP) Dependent Racemases Proposed mechanism for PLP-dependent alanine racemase H CH3 COOCOO- C + NH PLP CH3 H C Keq ~ 1 + 3 L-Ala NH3 D-Ala PLP PLP :B B H COO- H CH3 C + =O CH3 =O CH3 C + NH OH 3PO COO- COO- H + NH NH OH 3PO C =O OH 3PO : + Scheme 9.4 N H CH3 CH3 N H quinonoid intermediate Usually, a one-base mechanism + N H CH3 PLP was a coenzyme for decarboxylases (break C-COOH bond) and now for racemases (break C-H bond) How can PLP enzymes catalyze selective bond cleavage? Stereochemical Relationship Between the -Bonds Attached to C and the p-Orbitals of the -System in a PLP-Amino Acid Schiff Base H R -OOC NH NH Figure 9.2 PLP all sp2 + p atoms The -bond that is parallel to (overlapping with) the p-orbitals will break (C-H in this case) Dunathan Hypothesis for PLP Activation of the Bonds Attached to C in a PLP-Amino Acid Schiff Base The rectangles represent the plane of the pyridine ring of the PLP. The angle of viewing is that shown by the eye in Figure 9.2. pyridine ring of PLP COO- + H R C N CH COO- A + N CH + C R H R B C N CH -OOC H C Figure 9.3 The -charge stops free rotation, which results in selective bond cleavage Other Racemases Reaction catalyzed by mandelate racemase HO Ph H H CO2- Ph R-mandelate OH CO2- S-mandelate Scheme 9.5 No internal return in either direction With (R)-mandelate no -H exchange with solvent With (S)-mandelate there is exchange with solvent A Two-base Mechanism for Mandelate Racemase that Accounts for the Deuterium Solvent Exchange Results. Lys-166 acts on the (S)-isomer, and His-297 acts on the (R)-isomer OH OH : 166Lys A ND2 O (S) ND 166Lys O- Ph 297His D H H + N D D O- Ph OH O- Ph O Mg2= solvent exchange O Mg2+ ND 297His N : N H B HO H OH HO O- Ph OPh O (R) Mg2+ Scheme 9.6 H O- Ph O Mg2+ no solvent exchange O H297N mutant is capable of exchanging the -H of the S-isomer, but not the R-isomer H297N Mutant Capable of Elimination of HBr from (S)-9.5, but not from the (R)-isomer Elimination of HBr from (S)-p-(bromomethyl)mandelate, catalyzed by the H297N mutant of mandelate racemase NH2 Lys-166 H OH COO- Br 9.5 -HBr OH O COO- COO9.6 Scheme 9.7 K166R mutant catalyzes elimination of HBr from the (R)-isomer, but not from the (S)-isomer Epimerases Peptide epimerases Mechanism 1 Elimination/addition (dehydration-hydration) mechanism for peptide epimerization -OH OH OH O AcNH-Gly-Leu H Phe-Ala-OH N H B O H Phe-Ala-OH N H AcNH-Gly-Leu B H O :B O H O Phe-Ala-OH N H AcNH-Gly-Leu O 9.7 :B H B H B Scheme 9.8 H O AcNH-Gly-Leu OH OH H O Phe-Ala-OH N H AcNH-Gly-Leu Phe-Ala-OH N H O B O H With 18O 18O in the Ser OH group, no loss of as H2 Therefore, mechanism 1 is unlikely. 18O :B -Cleavage Mechanism for Peptide Epimerization Mechanism 2 O H O O AcNH-Gly-Leu :B H O B N H AcNH-Gly-Leu H O H H Phe-Ala-OH H Phe-Ala-OH N H AcNH-Gly-Leu 9.8 H Phe-Ala-OH N H O O OH O H :B B Scheme 9.9 10 mM NH2OH has no effect on product formation Therefore, mechanism 2 is unlikely. Deprotonation/Reprotonation Mechanism Mechanism 3 B B O AcNH-Gly-Leu H OH B O H N H AcNH-Gly-Leu Phe-Ala-OH N H AcNH-Gly-Leu H H B H H O O O HO B H H Phe-Ala-OH B: OH Phe-Ala-OH N H O :B B Scheme 9.10 In D2O D is incorporated into product, not substrate (short incubation; monitored by electrospray ionization mass spectrometry) Deuterium isotope effect for [-2H]-peptides in the L- to D-direction is different from that in the D- to L-direction (two-base mechanism) These results are consistent with mechanism 3. Epimerization with Redox Catalysis Proposed mechanism for dTDP-L-rhamnose synthase-catalyzed conversion of dTDP-4-keto-6-deoxy-D-glucose (9.9) to dTDP-L-rhamnose (9.10) H CH3 B+ H O O H OHH NADPH HO H H OdTDP H two different enzymes B+ O H B+ H CH3 O H H H OdTDP OH reductase :B CH3 H OH 9.10 9.9 epimerase B: Scheme 9.11 OdTDP OH OH B: O H H CH3 H OH B+ O H H H OH H3C O H H OdTDP OH H B+ H O H O H H B: OdTDP OH H O H H O H H CH3 H OdTDP OH O OH OH NH2 : N R C-H cleavage at C-3 and C-5 show kinetic isotope effects (3.4 and 2.0, respectively) In 2H2O 2H incorporation at both C-3 and C-5 Partial exchange gives only C-3 proton exchange, never only C-5 proton exchange (ordered sequential mechanism) UDP-Glucose 4-Epimerase OH O OH OH O UDP OH 9.11 UDP-glucose OH HO O OH O UDP OH 9.12 UDP-galactose In H218O, no incorporation of 18O into product No change in oxidation state, but is deprotonation/reprotonation reasonable? Tritium is incorporated from NAD3H into a derivative of the suspected intermediate of the UDP-glucose 4-epimerasecatalyzed reaction The enzyme requires NAD+; no exchange with solvent without OH CH3 reverse reaction CH3 O E•NAD3H + O HO OH O OH O dTDP OH 3H O dTDP OH 9.13 proposed intermediate Scheme 9.12 Proposed Mechanism for Reaction Catalyzed by UDP-Glucose 4-Epimerase NAD+ H OH O O O OH O UDP OH O H OH OH B H HO OH OH O UDP OH NAD H 9.14 O H NAD+ O UDP OH :B Scheme 9.13 Evidence for 9.14: incubate enzyme with UDPgalactose,quench with NaB3H4. 3H at C-4 of both UDP-glucose and UDP-galactose Mechanism to Account for Transfer of Hydrogen from the Top Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate OH H2N OH O O H R N OH O H H2N O UDP O H H O B OH O R N H Scheme 9.14 OH H HO HO HO :B -NAD+ O UDP OH O HO O UDP Mechanistic Pathway for the GDP-D-mannose-3,5epimerase-catalyzed Conversion of GDP-D-mannose (9.15) to GDP-L-galactose (9.18) No change in oxidation state, but NAD+ required OH H OH O OH NADH O O OH OH O O GDP OH 9.16 9.15 H NAD+ O OHOH OH Scheme 9.15 O OH O GDP O GDP OH OH OH NAD+ OH 9.18 NADH O O OH OH O GDP O GDP OH 9.17 [1,2]-H Shift Reaction catalyzed by aldose-ketose isomerases Lobry de Brun-Alberda von Ekenstein Reaction H CHO CH2OH C C R 9.19 Scheme 9.16 OH R 9.20 O Two Mechanisms Mechanism 1 cis-Enediol mechanism for aldose-ketose isomerases cis-enediol H B: H* C O H B B OH R (2R) B OH O H R (2R) Scheme 9.17 B H* H R C OH O :B OH O H B H B R 9.22 H* R OH R O : H* H B H pro-R * H C suprafacial transfer of H 9.21 B: : C H re OH C :B H* C H R re O B H H Mechanism 2 Hydride transfer mechanism for aldose-ketose isomerases H H C O H* C O H H R B :B *H H C O C O R H :B B Scheme 9.18 Partial incorporation of solvent observed inconsistent with hydride mechanism [1,3]-H Shift Enolization Reaction catalyzed by phenylpyruvate tautomerase R R O OH CO2HS HR R = H or OH Scheme 9.19 CO2HS removes pro-R hydrogen Two Conformers Possible Conformations of phenylpyruvate that would form Z- and E-enols by phenylpyruvate tautomerase O H B OH anti CO2- CO2Z HS HR B: CO2- CO2- syn O HS HR H B B: Scheme 9.20 OH E To Test for Favored Conformation R R CO2- F R 9.24 CO2- CO2- F CO29.23 R 9.25 9.26 favored inhibitors Therefore syn geometry to E enol most likely Allylic Isomerizations Mechanism 1 Carbanion mechanism for allylic isomerases H B: Scheme 9.21 B H H This H could come from the substrate (if no solvent exchange) Mechanism 2 Carbocation mechanism for allylic isomerases H H H B+ Scheme 9.22 H H B: This H comes from solvent, not from the substrate Mechanism 3 [1,3]-Sigmatropic hydride shift mechanism for allylic isomerases H H Scheme 9.23 Unlikely -- [1,3]-hydride shift is allowed antarafacial, which is geometrically impossible Carbanion Mechanism Reaction catalyzed by 3-oxo-5-steroid isomerase Scheme 9.24 O O 1 2 3 4 O H (D) 5 6 H H 9.27 O H H (D) H 9.28 Principal reaction transfers 4-H to 6-position; therefore suprafacial Eliminates carbocation mechanism and [1,3] hydride shift Evidence for an Enol Intermediate in the Reaction Catalyzed by 3-Oxo-5-steroid Isomerase O O enzymatic O O 9.32 9.31 O O non enzymatic pH 4.5 HO 9.33 enzymatic 9.32 Scheme 9.26 9.31 (90%) + O 9.34 10% Kinetic Competence of Enol Further evidence for an enol intermediate in the reaction catalyzed by 3-oxo-5-steroid isomerase O HO 9.35 O O O O 9.36 Scheme 9.27 same rates 9.37 From Site-directed Mutagenesis, Tyr-14 is the Acid and Asp-38 the Base Asp-38 O O H H O Tyr-14 suprafacial H O H O H O Tyr-14 orthogonal (favored) Tyr-14 antarafacial 9.38 from NOE studies Reactions Designed to Investigate the Function of Tyr-14 at the Active Site of 3-oxo-5-steroid Isomerase To probe the function of Tyr-14 OH Uv spectrum bound to enzyme is same as neutral amine. OH + H+ + H2N Therefore Tyr-14 does not protonate C-3 carbonyl H3N 9.39 O O - H+ -O HO 9.40 Scheme 9.28 Structure bound to enzyme even at low pH (pKa of the phenol must be very low). Therefore Tyr-14 H bonds to dienolate Carbanion Mechanism Mechanism for suprafacial transfer of the 4-proton to the 6-proton of steroids catalyzed by 3-oxo-5-steroid isomerase O H _ O O O O O 2H H H H COO- O Scheme 9.29 O COO 2H Tyr-14 Tyr-14 O 2H H COOAsp-38 Asp-38 Asp-38 Tyr-14 H Asp-99 Located Adjacent to Tyr-14 One mechanism for the function of Asp-99 in the active site of 3-oxo-5-steroid isomerase O Tyr14O H O Tyr14O Tyr14O H O Asp99 COO O H O H O H H O 38Asp Scheme 9.30 Asp99 COO H O H O O 38Asp H H H Asp99 COO O H O 38Asp H Crystal structure with equilenin bound is consistent with Asp-99 and Tyr-14 both coordinated to oxyanion O HO 9.41 equilenin 4-Oxalocrotonate Tautomerase CO2- CO2- O CO29.42 CO2- OH CO29.43 O CO29.44 Scheme 9.32 From deuterated substrates, substrate analogues, and reactions run in D2O, 9.42 to 9.44 is suprafacial (one-base mechanism) Carbocation Mechanism Reaction catalyzed by isopentenyl diphosphate isomerase O O O P O P OOO9.45 isopentenyl diphosphate O Mg++ O O P O P OOO9.46 dimethylallyl diphosphate Scheme 9.33 No exchange of solvent into substrate, only into product One base mechanism Evidence for a Carbocation Mechanism OP2O63- OP2O63- CF3 HN+ 9.49 9.48 rate is 1.8 10-6 times OP2O63- Ki = 14 pM transition state analogue inhibitor Proposed Mechanism for Isopentenyl Diphosphate Isomerase OPP B 2H OPP 2H OPP H 2H B: Scheme 9.35 Aza-allylic Isomerization H +NH +NH H Scheme 9.36 PLP-dependent Aminotransferase Reaction catalyzed by L-aspartate aminotransferase O H -OOC CH2 14C COO- + 15 NH + 3 Scheme 9.37 CH3 13C COO- H218O H -OOCCH 14C 2 18O COO- + CH3 13C COO- 15NH + 3 First Half Reaction Catalyzed by Aspartate Aminotransferase B: H NH OH =O PO 3 -OOC H -OOC N H 14C COO- 14C see Scheme 8.39 15NH 15NH 2 slow step OH =O PO 3 9.50 COO- N H aldimine -OOC B H 14C COO- 18O -OOC 14C 15NH COO- 15NH 9.51 H218O 15NH 3 OH =O PO 3 .. N H quinonoid Scheme 9.38 COO- -OOCCH 14C 2 OH =O PO 3 OH =O PO 3 N H N H 9.52 PMP Second Half Reaction Catalyzed by Aspartate Aminotransferase H B 15NH 3 CH3 O OH =O PO 3 CH3 N H 13C B: COO- OH =O PO 3 N H H H CH3 13C COO- CH3 OH =O PO 3 N H 13C COO- 15NH 15NH 15NH H 9.53 9.52 13C COO- CH3 COO- 13C 15NH 3 NH2 OH =O PO 3 NH N H OH =O PO 3 N H Scheme 9.39 This is the reverse of the mechanism in Scheme 9.38 Crystal structures of: • native enzyme with PLP bound • substrate reduced onto PLP • enzyme with PMP bound All are consistent with mechanisms in Schemes 8.39 and 9.38 Evidence for Quinonoid Intermediate OH COO- -OOC + NH OH COO- -OOC + NH3 9.54 pseudosubstrate OH =O PO 3 N H 9.55 quinonoid form observed at 490 nm Stereochemistry of Proton Transfer in the First Step Catalyzed by Many PLP-dependent Aminotransferases -H is transferred to the CH2 of PMP suprafacially; therefore one-base mechanism -2H removed from si-face and delivered to pro-S CH2 of PMP B B: H N -O H3C 2H R H OPO3 N H -OOC 2H COO- 9.56 -O = H3C H N N B: -OOC R H H N -O OPO3 = R 2H pro-S H H3C OPO3= N H H H2O Scheme 9.40 H2N R -OOC O -O H3C H OPO3= N H 2H 9.57 Cis-Trans Isomerization Reaction catalyzed by maleylacetoacetate isomerase COO- GSH -OOC COOO O 9.58 COOO O 9.59 Scheme 9.41 GSH acts as a coenzyme, not as a reducing agent No 2H incorporated into substrate or product from 2H2O Proposed Mechanism for the Reaction Catalyzed by Maleylacetoacetate Isomerase GS COO- GS COO- -OOC COOO O Scheme 9.42 -OOC SG COOO O COO- COOO O O O Phosphate Isomerization Reaction catalyzed by phosphoglucomutases OPO3= O HO HO OH OH 9.66 only -anomer binds Scheme 9.45 HO HO OH O OH 9.67 OPO3= Native State of Enzyme is Phosphorylated Proposed mechanism for the reaction catalyzed by phosphoglucomutases B H Ser O O B 32 P OO OH :B O HO HO OH 9.67 Scheme 9.46 Ser O OPO3= tightly bound H Ser O PO3= O32PO3= O32PO3= O HO HO 9.68 O OOH O P O- B H HO HO O Overall retention of configuration at phosphate Double inversion Shown as associative, but could be dissociative OH 9.66 OH Model Reaction for a Dissociative Mechanism of Phosphomutases S SO 18O- P O 18O- P H O O O OH OH NO2 NO2 solvent cage Scheme 9.47 S 18OP O O NO2 ~ 40% retention