Mechanism-based enzyme inactivation using an allyl sulfoxide-allyl sulfenate ester rearrangement

Abstract

2-Amino-4-chloro-5-(p-nitrophenylsulfinyl)pentanoic acid (1) was synthesized and shown to induce mechanism-based inactivation of 2 pyridoxal phosphate dependent enzymes: [Salmonella typhimurium] cystathionine .gamma.-synthetase [EC 4.2.99.9], which catalyzes a .gamma.-replacement reaction in bacterial methionine biosynthesis; and [Pseudomonas ovalis] methionine .gamma.-lyase, [EC 4.4.1.11], which catalyzes a .gamma.-elimination reaction in bacterial methionine breakdown. The inactivations are irreversible and display saturation kinetics. Each enzyme incorporates roughly 1 mol of tritium/mol of enzyme monomer when inactivated by 2-amino-4-chloro-5-(p-nitro[3H]phenylsulfinyl)pentanoic acid, confirming that the modification of each protein is covalent and stoichiometric. Substoichiometric labeling (0.12 mol of tritium/mol of enzyme monomer) is given when methionine .gamma.-lyase is fully inactivated by 2-amino-4-chloro-5-[3H]-5-(p-nitrophenylsulfinyl)pentanoic acid. Both enzymes, inactivated by 1, are susceptible to reactivation by thiols. Inactivated cystathionine .gamma.-synthetase recovers 25% of its catalytic activity upon incubation with excess dithiothreitol, while methinonine .gamma.-lase is 100% reactivated by dithiothreitol, mercaptoethanol and mercaptopropionate. Reactivation generates p-nitrophenylthiolate anion, which forms, in the case of methionine .gamma.-lyase, stoichiometrically with enzyme reactivated. Both enzymes are protected from inactivation by 1 in the presence of thiols, which simultaneously generates p-nitrophenylthiol. In the presence of dithiothreitol, the protection reaction gives p-nitrophenylthiol production with pseudo-1st-order kinetics. 2-Amino-4-chloro-5-(p-tolylsulfinyl)pentanoic acid and 2-amino-4-(p-nitrophenylsulfinyl)-5-chloropentanoic acid, the reverse regioisomer of 1, were also prepared and gave no evidence of inactivation of either enzyme. A novel form of suicide inactivation may be indicated (Scheme II) wherein .beta.-carbanion-assisted .gamma.-halide elimination generates an allyl sulfoxide-enzyme-pyridoxal adduct which undergoes spontaneous 2,3-digmatropic 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.