The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 8 Decarboxylation

Scheme 8.1 Decarboxylation Reactions Driving force for decarboxylation

Scheme 8.2 Decarboxylation is accelerated in acid Decarboxylation of  -Keto Acids

Scheme 8.3 Strong acids are needed to protonate carbonyls (pK a -7) Cyclic Transition State for Decarboxylation of  -Keto Acids

Scheme 8.4 Protonation of Imines (pK a about +7) is Easy Amine-catalyzed decarboxylation of  -keto acids

Scheme 8.5 Reaction Catalyzed by Acetoacetate Decarboxylase

Scheme 8.6 Schiff Base Mechanism Suggests a Schiff base mechanism In D 2 O, D is incorporated into acetone pK a of Lys-115 is 5.9; adjacent to Lys-116, which lowers pK a by about 4.5 pK a units Fate of the ketone oxygen in the reaction catalyzed by acetoacetate decarboxylase Why?

Scheme 8.7 after aminoethylation it is active inactive mutant Lys-116 mutants are still active (but less than WT) pK a of Lys-115 in Lys-116 mutants is >9 Aminoethylation of K116C - lowers pK a of K115 back to 5.9 Reaction of the K115C Mutant of Acetoacetate Decarboxylase with 2-Bromoethylamine

Scheme 8.8 Schiff Base Mechanism Proposed mechanism for acetoacetate decarboxylase

Scheme 8.9 Test for Schiff Base Mechanism NaBH 4 reduction during the reaction catalyzed by acetoacetate decarboxylase isolated

Scheme 8.10 Metal Ion-catalyzed Mechanism Alternative to Schiff base mechanism With 14 C substrate + NaBH 4 no loss of carbonyl oxygen no 14 C protein

Scheme 8.11 inversion of stereochemistry Proposed Mechanism for the Decarboxylation of (S)-  -acetolacetate (8.11) Catalyzed by  -Acetolactate Decarboxylase

Scheme 8.15  -Hydroxy Acids isocitrate oxalosuccinate Oxalosuccinate is not detected. Also no partial reactions, but it is presumably formed Proposed mechanism for the isocitrate dehydrogenase-catalyzed conversion of isocitrate (8.17) to  -ketoglutarate (8.19)  -ketoglutarate

Scheme 8.16 Not All Decarboxylations Need Schiff Base or M 2+ 6-phosphogluconate Experiments? ribulose 5-phosphate Reaction catalyzed by phosphogluconate dehydrogenase

Scheme 8.17 Proposed Mechanism for the Reaction Catalyzed by Phosphogluconate Dehydrogenase

Scheme 8.18 Improbable decarboxylation of  -keto acids  -Keto Acids

Scheme 8.19 Cofactor Required thiamin (vitamin B1) thiamin diphosphate coenzyme vitamin Diphosphorylation of thiamin

Abbreviated Form for TDP

Scheme 8.20 exchangeable in neutral D 2 O at room temperature pK a estimated Resonance Stabilization of Thiazolium Ylide

Scheme 8.21 Without N-1 N or N-4 NH 2 it is not active Without N-3 N it is active Proposed Mechanism for Autodeprotonation of C-2 of Thiamin Diphosphate

Stable, but C-2 proton not very acidic 100 times more acidic, but does not catalyze  -keto acid decarboxylation and is easily hydrolyzed at pH 7 Ideal heterocycle

Scheme 8.26 Mechanism of Thiamin Diphosphate-dependent Enzymes pyruvate decarboxylase acetolactate synthase acetoin Nonoxidative decarboxylation of  -keto acids: (A) the reaction catalyzed by pyruvate decarboxylase, (B) the reaction catalyzed by acetolactate synthase

Scheme 8.27 Benzoin Condensation Chemical model for formation of 8.45

Scheme 8.29 nucleophile electrophile catalyst Mechanism for the Benzoin Condensation

Scheme 8.30 like - CN electrophile nucleophile Proposed Mechanism for the Reaction Catalyzed by Acetolactate Synthase catalyst

Scheme 8.33  -KG succinyl-CoA Multienzyme complexes 5 different coenzymes involved Examples of Oxidative Decarboxylation of  -Keto Acids

Scheme 8.34  -Keto Acid Dehydrogenase pyruvate decarboxylase dihydrolipoyl transacetylase lipoic acid TDP reduced lipoic acid acetyl lipoamide Proposed mechanism for the reaction catalyzed by dihydrolipoyl transacetylase -CO 2 dihydrolipoyl dehydrogenase FAD NAD + lipoic acid

Coenzyme A

Scheme 8.35 Alternative Proposed Mechanism for the Reaction Catalyzed by Dihydrolipoyl Transacetylase

Scheme 8.37 dihydrolipoyl dehydrogenase Proposed Mechanism for the Reaction Catalyzed by Dihydrolipoyl Dehydrogenase

Scheme 8.38 Amino Acid Decarboxylation covalently bound via Schiff base to a Lys residue pyridoxine (vitamin B 6 ) Pyridoxal 5-phosphate (PLP) coenzyme Conversion of pyridoxine to pyridoxal 5-phosphate (PLP)

Scheme 8.39 The First Step Catalyzed by All PLP-dependent Enzymes, the Formation of the Schiff Base between the Amino Acid Substrate and PLP

Scheme x faster than Schiff Base Formation Increases the Electrophilicity of the Carbonyl (A) Reaction of an amine with an imine (B) Reaction of an amine with an aldehyde

Scheme 8.41 neutralizes acidic conditions increases intracellular pressure Reaction Catalyzed by PLP Decarboxylases (different enzymes for different amino acids)

Scheme 8.42 To Provide Evidence for a Schiff Base with an Active Site Lysine Residue Reduction and hydrolysis of PLP enzymes.

If Substrate Is Added before NaBH 4

Scheme 8.43 No 18 O from H 2 18 O Found in CO 2 Incorrect hydrolytic mechanism for PLP-dependent enzymes

Scheme 8.44 electron sink to stabilize anion stereospecific incorporation of proton Proposed Mechanism for PLP-dependent Decarboxylases

Scheme 8.45 PLP and PQQ enzymes have absorbance >300 nm Pyruvoyl enzymes - no absorbance >300 nm PLP and PQQ enzymes do not give the products shown above Proposed mechanism for pyruvoyl-dependent decarboxylases (amino acids) Pyruvoyl-Dependent Decarboxylases - Identification and Differentiation Differences

Scheme 8.46 S-adenosylmethionine (SAM) Inactivation of S-Adenosylmethionine Decarboxylase by its Substrate inactivation

Scheme 8.47 prohistidine decarboxylase both 18 O end up here Biosynthesis of the Active-site Pyruvoyl Group of Histidine Decarboxylase Pathway b is valid only if the hydroxide released is the same one that hydrolyzes the amide bond.

Scheme 8.48 Proposed addition/elimination for orotidine 5- monophosphate decarboxylase Other Decarboxylations

Scheme 8.49 Proposed Zwitterion Mechanism for Orotidine 5-Monophosphate Decarboxylase

Scheme 8.50 Model Study for the First Mechanism Model reactions for the addition/elimination mechanism for orotidine 5-monophosphate decarboxylase

incubate with enzyme - no rehybridization by 13 C NMR no secondary deuterium isotope effect Support for the Second Mechanism (actually, disproof of the first mechanism)

X = Br, Cl inhibitors X = F substrate excellent substrate no rehybridization More Evidence Against the First Mechanism

When R = COOH OMP decarboxylase is the most proficient enzyme known k cat = 39 s -1 k non = 2.8  s -1 (nonenzymatic) k cat /k non = 1.4  (rate enhancement)

protonation at O-2 original proposal protonation at O-4 based on calculations Two atoms of Zn 2+ in active site - may stabilize negative charge Rationale for Direct Decarboxylation

Scheme 8.51 Decarboxylative elimination Reaction Catalyzed by Mevalonate Diphosphate Decarboxylase

Both are poor substrates Destabilize a carbocation intermediate

Very Potent Inhibitor - TS ‡ Analogue Inhibitor mimics a carbocation

Scheme 8.52 Proposed Carbocation Mechanism for Mevalonate Diphosphate Decarboxylase