Mechanism-Based Enzyme Inactivation Using an Allyl Sulfoxide-Allyl Sulfenate Ester Rearrangement.

2-Amino-4-chloro-5-(-nitrophenylsulfinyl)pentanoic acid (1) has been synthesized and shown to induce mechanism-based inactivation of two pyridoxal phosphate dependent enzymes: (1) cystathionine γ-synthetase, which catalyzes a γ-replacement reaction in bacterial methionine biosynthesis; and (2) methionine γ-lyase, which catalyzes a γ-elimination reaction in bacterial methionine breakdown. The inactivations are irreversible and display saturation kinetics. Each enzyme incorporates roughly 1 mol of tritium per mol of enzyme monomer when inactivated by 2-amino-4-chloro-5-(-nitro[H]phenyl-sulfinyl)pentanoic acid (), confirming that the modification of each protein is covalent and stoichiometric. Substoichiometric labeling (0.12 mol of tritium per mol of enzyme monomer) is given when methionine γ-lyase is fully inactivated by 2-amino-4-chloro-5-[H]-5--nitrophenylsulfinyl)pentanoic acid (). Both enzymes, inactivated by , are susceptible to reactivation by thiols. Inactivated cystathionine γ-synthetase recovers 25% of its catalytic activity upon incubation with excess dithiothreitol, while methinonine γ-lyase is 100% reactivated by dithiothreitol, mercaptoethanol, and mercaptopropionate. Reactivation generates -nitrophenylthiolate anion, which forms, in the case of methionine γ-lyase, stoichiometrically with enzyme reactivated. Both enzymes are "protected" from inactivation by in the presence of thiols, which simultaneously generates -nitrophenylthiol. In the presence of dithiothreitol, the protection reaction gives -nitrophenylthiol production with pseudo-first-order kinetics. 2-Amino-4-chloro-5-(-tolylsulfinyl)pentanoic acid () and 2-amino-4-(-nitrophenylsulfinyl)-5-chloropentanoic acid (), the reverse regioisomer of , have also been prepared and give no evidence of inactivation of either enzyme. The data are taken to indicate a novel form of suicide inactivation (Scheme II) wherein β-carbanion-assisted γ-halide elimination generates an allyl sulfoxide-enzyme-pyridoxal adduct () which undergoes spontaneous 2,3-sigmatropic rearrangement to an electrophilic allyl sulfenate ester (). The latter is then captured by an enzymic nucleophile to give an inactive enzyme , which may be a mixed disulfide or, less likely, a sulfenamide.