The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 6

The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 6

The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 6 Substitutions SN1 Reactions catalyzed by farnesyl diphosphate synthase -PPi + PPO 6.1 isopentenyl DP PPO geranyl diphosphate PPO 6.2 6.3 dimethylallyl DP -PPi PPO farnesyl diphosphate PPO 6.4 Scheme 6.1 Hammett study supports carbocation intermediate PPO F 6.5 Km same as geranyl DP, but kcat 8.4 10-4 times that with geranyl DP Therefore, it binds as well as geranyl DP, but is converted to product at a much slower rate, supporting an electron-deficient intermediate

(such as a carbocation). Model Studies to Test Mechanism 1. Solvolysis (carbocation mechanism) rate with X = F is 4.4 x 10-3 times rate with X = H 2. SN2 rate with X = F is 2 x faster than when X = H O H3C S O O X 6.7 The enzymatic reaction is 8.4 x 10-4 times slower when X = F compared to X = H Therefore carbocation mechanism Further Support for Carbocation Mechanism CHF2 CH2F PPO PPO 6.8 Relative rate 1.75 10-2 CF3 PPO 6.9 6.10 1.90 10-6 3.62 10-7 compared with geranyl DP (CH3) Km values similar to geranyl DP Rates correlate with nonenzymatic solvolysis for fluorinated methanesulfonates relative to geranyl DP (carbocation mechanism) Carbocation Mechanism (SN1) for

Farnesyl Diphosphate Synthase + PPO R R 6.11 R = Me (6.2) R = C5H11 (6.3) + PPO + PPi R + PPO H B: Scheme 6.2 R PPO R Stereochemistry of Farnesyl Diphosphate Synthase syn addition/elimination PPO 6.21 si PPO B: HR HS

6.20 Figure 6.1 Sesquiterpenes Biosynthesized from Farnesyl DP Reaction catalyzed by pentalenene synthase 309His 309His N NH NH N H H 6.22 PPO humulene 309His Mg2+ N 309His NH N H H H H NH H H

6.24 pentalenene 6.23 Scheme 6.5 From the Crystal Structure of Pentalenene Synthase Stabilization of carbocation intermediates by active-site phenylalanine and asparagine residues cation- interaction O NH2 C 219Asn Phe77 Figure 6.2 carbocation stabilization SN1/SN2 Reaction catalyzed by phosphorylases O R O R' + -O P OOH Scheme 6.6 O RO P

O- O- + R'OH Reaction Catalyzed by Disaccharide Phosphorylases OH OH another sugar O OH + HO 18O HO R 6.25 Scheme 6.7 O OH Pi OPO3= + HO HO 6.26 H18OR 6.27 Stereochemistry of the Reactions Catalyzed by Disaccharide Phosphorylases

cellobiose phosphorylase m ltose hoshorylse sucrose hoshorylse C-1 Configuration of Disaccharide C-1 Configuration of Phosphorylated Product inversion retention Two Mechanisms for Inversion SN2 versus stereospecific SN1 reaction OH SN2 OH OH HO O HO SN1 S 2 N -OPO OR Scheme 6.8 3 OH HO H- O OPO3H-

HO OH + SN1 OH HO O -OPO H3 HO No partial exchange reactions with cellobiose or maltose phosphorylases (consistent with SN2) With sucrose phosphorylase, [14C]fructose is incorporated into sucrose in presence of unlabeled sucrose and in absence of Pi Suggests double SN2 displacement Covalent Catalysis sucrose phosphorylase OH OH O HO HO HO O HO O OH HO glucosyl fructosyl 6.28 C in glucosyl part

gives 14C-protein (quench at low pH) 14 C in fructosyl part gives no 14C-protein 14 Again consistent with a double displacement mechanism Experiments to Identify Active Site Residue (X) 1. [14C] glucosyl enzyme OH O HO HO MeOH 6.29 (R = H), not 6.29 (R = Me) (glucose) OH O X HO OH HO OR OH 6.29 2. [14C] glucosyl enzyme very sensitive to base 3. [14C] glucosyl enzyme NH2OH 6.29 (R = H) + O NHOH Therefore X is Glu or Asp

Disaccharide Phosphorylase Reactions Involving an Active-site Carboxylate OH OH HO .. O HO B+ O O- SN2 OH SN2 OH HO OR -OPO H3 SN2 a SN1 OH + OH Scheme 6.9 O HO 6.31 H HO

O O O a - O HO 6.32 OH O b b -OPO H3 OH HO O HO OPO3H- Two Mechanisms for Reactions Catalyzed by -Glycosidases--Hydrolysis of Disaccharides acid HO A Scheme 6.10 OH O HO O -ROH R

HO H OH -O OH O O H O HO HO HO O OH HO O SN2 (inversion) O (General acid/base mechanism) O base acid B HO HO H O OH O HO OR -O O -ROH HO

HO OH O HO O O H O OH O HO HO O OH HO O OH HO nucleophile Two active site carboxylic acids -O O double SN2 O (retention) (Covalent) Mutation to Ala: kcat 107-fold lower Add in N3- to replace the carboxylate nucleophile: kcat only 102-fold lower (-azide forms) Differentiation of SN2 from SN1 for -Glycosidases OH O HO HO F

6.33 X=F X O or NO2 really good leaving groups NO2 more electronegative than OH destabilizes an oxocarbenium ion intermediate SN1 reaction slower than glycoside SN2 reaction faster than glycoside Covalent adduct stabilized Reaction of 6.33-Inactivated -Glucosidase with 6.35 HO HO OH O OH O + F O O HO HO OH O OH HO

HO 6.35 6.34 after isolation (Glu-358) Ph HO HO O HO Ph OH F 6.36 OH O OH O X Scheme 6.11 F 6.33 2nd step must be 6.36 formed from 6.34 at same rate as from rds 6.33 Therefore 6.34 is a kinetically competent intermediate, consistent with SN2 mechanism followed by SN1 Both SN2- and SN1-like Character of -Glucosidase H B HO HO

OH O OH O H OR B SN2-like HO HO HO O substitution of Glu-358 by Asn or Gln - inactive by Asp - 2500x slower OH O O OH OR HO HO O B H HO O O O H

O SN1-like H B HO HO OH O OH O Scheme 6.12 H OH O B HO HO OH OH O+ HO O O SN2 Two mechanisms for epoxide hydrolase General base mechanism A Enz B: O H EnzBH OH OH + HO Nucleophilic (covalent) mechanism

O B Enz B O O Enz O OH O O Enz BH+ B: H OH OH O + HO Scheme 6.14 Single-turnover experiment in H218O - no 18O in glycol Enzyme labeled with 18O in active site Asp gives 18O glycol Consistent with covalent catalytic mechanism Further Evidence for Ester Linkage Covalent intermediate isolated during reaction catalyzed by epoxide hydrolase H+ O 3 H OH 3H CO2Me O

O- O 6.38 Asp333 N OH 3H CO2Me O H OH Asp 6.39 quenched (AcOH) 333 precipitated (acidic acetone) NH OH 3H OH HO OH 3H LiAlH4 O 6.40 Scheme 6.15 CO2Me HO CO2Me O Asp333

isolated NaOH OH 3H CO2H HO 6.41 A Catalytic Antibody-catalyzed 6-Endo-tet Ring Closure Baldwins rules predict 5-exo-tet a HO 6-endo-tet O Ar 6.43 O b b Ar obtained with a catalytic antibody (anti-Baldwin product) Scheme 6.16 a :O b H 6.42 HO 5-exo-tet H

a Ar H O 1.8 kcal/mol lower in energy in solution SN2 Reaction catalyzed by isochorismate synthase COO- COO- O COO- OH 6.44 Scheme 6.18 isochorismate synthase Mg++ H218O 18OH O 6.45 COO- SN2 Mechanism for Isochorismate Synthase H COO- 18O :B H -OOC

O COO- O COO- OH B+ H 6.44 H 2+ Mg O18 H 6.46 Scheme 6.19 18OH O H O COO- all axial conformation COO- Reaction Catalyzed by Anthranilate Synthase CO2- CO2NH3 O CO2-

-H2O CO2NH3 O OH 6.44 Scheme 6.20 CO2- 6.47 synthesized kinetically competent intermediate 6.48 NH3+ Reaction Catalyzed by p-Aminobenzoic Acid (PABA) Synthase synthesized kinetically competent Scheme 6.21 CO2- CO2- CO2- NH3 O OH 6.44 CO2- -H2O O CO2- +NH 3 6.49

reaction different from others NH3+ 6.50 Synthesized as TS Mimics of the 3 Enzymes (in the all-axial conformation) isochorismate synthase CO2- CO2- OH O anthranilate synthase CO2- OH OH 6.51 6.52 CO2- + NH3 O PABA synthase OH CO2- O + NH CO2- 3 6.53 All 3 compounds competitive inhibitors of respective

enzymes; bind tightly to isochorismate and anthranilate synthases, but weakly to PABA synthase (different mechanism) Nucleophilic Aromatic Substitution (SNAr) Glutathione (GSH) COO- H N + H3N O O N H COO- SH 6.57 -glutamylcysteinylglycine SNAr Reaction catalyzed by glutathione S-transferase Tyr O Tyr GS Scheme 6.23 O H X O N O+ slow Y

H _ O X _ N O + GS GS NO2 fast + Y Hammett study r = +1.2 for GSH = +2.5 for -Glu-Cys rate X = F > X = Cl HX Y therefore carbanionic Glutathione S-transferase-Catalyzed Reaction of Glutathione with 1,3,5-Trinitrobenzene O2N NO2 N+ O O- GS GS- H NO2 O2N NO26.58

Scheme 6.24 observed spectroscopically Meisenheimer complex Electrophilic Substitution (Addition/Elimination Mechanism) Reaction catalyzed by 5-enolpyruvylshikimate3-phosphate (EPSP) synthase CO2- CO2+ =O 3PO OH OH + =O CO2- 3PO 6.61 6.60 shikimate-3-P =O 3PO O Pi CO2- OH 6.62 PEP EPSP

Scheme 6.29 Herbicide Glyphosphate (Roundup) inhibits EPSP synthase + -OOCCH NH CH PO = 2 2 2 3 6.63 4 Possible Mechanisms for EPSP Synthase 1) Concerted :B H H B+ ROH -O CH2 addition 2C + OPO3= RO H - Pi OPO3= RO elimination 6.62 CO2- CO2- B:

2) Stepwise H B+ -O 2C OPO3= ROH + -O 2C + RO OPO3= H OPO3= CO2- B: Scheme 6.30 (continued on next slide) B: H RO CH2 + CO2- 6.62 4 Possible Mechanisms for EPSP Synthase (continued) 3) Covalent-concerted (a) and 4) Covalent-stepwise (b)

X-O 2C H B+ :B H H2C b b X OPO3= stepwise _ CO2 a H B+ OPO3= RO H a B: H2C X X concerted H CH3 :B _ X CO2 RO Scheme 6.30 6.62 + _

CO2 ROH _ RO _ CO2 CO2 H2C X :B H _ CO2 RO H :B Isolated by Et3N Quench CO2CH3 =O PO 3 O OH OPO3= CO2- 6.64 Incubated with EPSP synthase kinetically competent intermediate Therefore not covalent mechanisms (3 or 4) Kinetic analysis indicates only one intermediate detected; therefore mechanism 1 proposed Evidence for Stepwise Mechanism 2 EPSP synthase-catalyzed reaction of

shikimate-3-phosphate and (Z)-3-fluoroPEP CO2- CO2- F CO2- F CH2F =O PO 3 OH =O 3PO OH 6.60 Scheme 6.31 COO- 6.65 =O PO 3 O OH 6.66 CO2OPO3= + Pi =O PO 3 O CO2- OH 6.67

isolated does not give 6.66 (reverse reaction) Not much carbocation character in the addition step, but high carbocation character in elimination step Carbocation Character in the Reaction Catalyzed by EPSP Synthase CO2- CO2CH3 =O PO 3 CO2- O OH OPO3= 6.68 Scheme 6.32 CH3 =O O 3PO OH 6.69 CO2- EPSP To Determine Stereochemistry of Tetrahedral Intermediate phosphonate (stable) CO2- CO2CH3

=O PO 3 O OH PO3= CO2- CH3 =O 3PO O OH CO2PO3= 6.71 6.72 Ki = 15 nM* (suggests this stereochemistry) Ki = 1130 nM To make a stable phosphate, put in an electron withdrawing group CH2F, CHF2, CF3 CO2- CO2X =O 3PO O OH 6.73 OPO3= CO2- X =O PO 3

O OH CO2OPO3= 6.74 more potent inhibitor (opposite stereochemistry as the phosphonate analogues) MurA (Bacterial cell wall peptidoglycan biosynthesis) Similar reaction to EPSP synthase Reaction catalyzed by uridine diphosphate-Nacetylglucosamine enolpyruvyl transferase (MurA) OH O O HO HO O NH O OH NH O O P O P O OO6.75 N O + O HO OH

=O PO 3 CO2 - -Pi HO O COO- O O O NH O NH O O P O P O OO6.76 N O O HO OH Scheme 6.34 opposite results Kinetics suggest tetrahedral noncovalent intermediate [14C]PEP or [32P]PEP gives labeled enzyme NMR with [2-13C]PEP shows phospholactyl enzyme adduct (kinetically competent) One Possible Mechanism for the Reaction Catalyzed by MurA covalent intermediate B

H OPO3= OPO3= CO2- CO2- UDP-GlcNAc X X- 6.77 phospholactyl enzyme kinetically competent Scheme 6.35 noncovalent intermediate HO O OH O - HN O-UDP CO2 O OPO3= 6.78 OH HO O CO2- O + Pi HN O-UDP O 6.79 Further Evidence for Covalent and Noncovalent Intermediates

Inactivation of MurA by (E)- and (Z)-3fluoroPEP OPO3= H OPO3= F or F 6.80 CO2- Scheme 6.36 - H CO2 FCH2 OPO3= CO2X 6.65 6.81 covalent (stable) -O C 2 OH O HO O OPO3= FCH2 NH O UDP Ac

6.82 noncovalent Kinetics suggest that 6.82 does not come from 6.81 Branching Mechanism More consistent mechanism for the reaction catalyzed by MurA H OPO3= H - H B CO2 +OPO = 3 H3C CO26.83 OPO3= X CO2- H3C - Pi RO: 6.84 X- CH3 ROH OPO3=

CO2- 6.86 covalent intermediate noncovalent intermediate CO2- H H H O+ R B- H CO2- H OR 6.79 6.85 Scheme 6.37 Determination of the Stereochemistry of the Reaction Catalyzed by MurA Scheme 6.38 H OPO3= H F From crystal structure [2R];

therefore ROH addition is 2-si (top) (2-re in PEP) OR 6.80 CO2- OPO3= H CO26.65 MurA ROH/D2O OR H CO2D F OPO3 6.87E H F D H [2R] OPO3 [2R] -UDP-GlcNAc O F CO2- D OR CO2-

6.87Z alkaline phosphatase -UDP-GlcNAc F OPO3= CO2D 3R 2R 6.87E F O CO2- D F 6.88E fluoropyruvate H 6.88Z pyruvate carboxylase fluorooxaloacetate Analyzed for H or D by 19F NMR -OOC (retention) O -OOC CO2- D F CO2- H

F malate Therefore addition of D is to 3-re dehydrogenase face (bottom), which is called si OH with PEP; addition of ROH is to fluoromalate -OOC -OOC H CO22-si (top), which is 2-re in PEP H D F F Anti addition 6.89E + O OH H fluoromalate CO26.89Z Stereochemistry of the Reaction Catalyzed by MurA HO O H B: OH O re re NH Ac O UDP H

OPO3= H COO- si anti addition HO O H OPO3= H H S H 115Cys Scheme 6.39 OH O COO- NH Ac O UDP syn elimination HO O H COO-

H S 115Cys S H 115Cys Not concerted HPO4= OH O NH Ac O UDP si Electrophilic Aromatic Substitution Friedel-Crafts reaction (alkylation) R' H R' X AlCl3 R Scheme 6.40 -X R' + R R Enzymatic Friedel-Crafts Reactions (alkylation in nature) COO- COOCH3

+ O H OPP CH3 8 B+ O H 8 H CH3 :B B: COOcoenzyme Q vitamin K other quinones 8 OH Scheme 6.41 CH3 Electrophilic Heteroaromatic Substitution porphobilinogen deaminase COOH HOOC H2N A = acetate P = propionate N

H 6.90 P A HO N H N H N H P P A P A A N H 6.91 porphyrins heme corrins coenzyme B12 1) E2' (1,6-elimination) Three Possible Mechanisms for the Reaction Catalyzed by Porphobilinogen Deaminase concerted P A

P A N NH3+ Nu N N H Nu H B 2) E1cB P A anionic P A Nu N NH3+ N NH3+ H B 3) E1 Scheme 6.43 cationic P A

NH3+ N H P A P A P A N N H H Substrate Analogues P A N NH2 CH3 6.92 H3C NH2 N H 6.93 substrate (but no tetrapyrrole formed-only tripyrrole) Therefore E2 and E1cB unlikely

P A P A F3C NH2 N H 6.94 excellent substrates F3C H3C OH N H P A P A OH 6.95 N H 6.96 not substrates Consistent with E1 mechanism Cation Mechanism Most Reasonable Carbocation mechanism for porphobilinogen deaminase

A A A P NH2 N NH2 H + H B N H P N H -NH3 P A A P N+ H NH2 P A +N H H P P 6.91 N+ H

H :B A N H NH2 B: HO P N H A Scheme 6.44 A N H P P A A N H P N H P A N H

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