Mechanism-based inactivation of L-methionine .gamma.-lyase by L-2-amino-4-chloro-4-pentenoate

Abstract

L-2-amino-4-chloro-4-pentenoic acid (L-ACP), an antibacterial amino acid produced by Amanita pseudoporphyria Hongo time dependently and irreversibly inactivates L-methionine {gamma}-lyase. The inactivation obeys biphasic pseudo-first-order kinetics and is carried out completely with a minimum molar ratio ((L-ACP)/(enzyme tetramer)) of 5. During the incubation of enzyme, 4.4-5.0 mol of chloride ions is formed per mole of tetramer enzyme. The tetrameric enzyme is labeled with 4 mol of DL-(2-{sup 14}C)ACP/mol. The authors have isolated {sup 14}C-labeled acetopyruvate and pyridoxamine 5'-phosphate from the ({sup 14}C)ACP-modified enzyme. The enzyme fully inactivated shows {lambda}{sub max} at 460 and 495 nm, which probably is derived from a conjugated pyridoximine parquinoid. The authors have proposed a mechanism which involves enzymatic dehalogenation from C{sub 4} of ACP to form a reactive allene. The allene is attacked by a nucleophilic amino acid residue at the active site. Analysis results of the thiol content of enzyme suggest that a cysteine residue is a possible nucleophilic residue covalently bound to the inactivator.