Abstract
The L-methionine γ-lyase of Pseudomonas putida contains 16 cysteinyl residues per mol of enzyme, which is composed of four identical polypeptide chains. Eight of these residues are buried inside the enzyme structure; they reacted with 5, 5'-dithiobis(2-nitrobenzoic acid) only after the enzyme was denatured. In the native enzyme, two of them were cyanylated with 2-nitro-5-thiocyanobenzoic acid with concomitant inactivation of the enzyme. Both the cyanylation and the inactivation proceeded through biphasic pseudo-first order kinetics. Cyanylation of about 2 mol of cysteine residues per mol of enzyme practically abolished the α, γ-elimination activity: the enzyme shows a half-of-the-sites reactivity. L-Norleucine, a competitive inhibitor, which binds to the active site, strongly retarded the reaction of the essential cysteine residue with 2-nitro-5-thiocyanobenzoic acid, and protected the enzyme from inactivation. The cyanylated enzyme was reactivated with 2-mercaptoethanol formed in situ from S-(β-hydroxyethyl)-L-homocysteine by its remaining activity. Added 2-mercaptoethanol was much less effective than that formed in situ. These suggest that the essential cysteine residue is at or near the active site. The cyanylated enzyme was substantially inactive (residual activity, 5%) for the α, γ-elimination, but 40% active for the α, β-elimination. Kinetic analyses revealed that the affinity of enzyme for the substrates, particularly for long straight-chain amino acids, was decreased greatly by cyanylation. The cyanylated cysteine residue was found to be located at a position of approximately 20% of the total length of the polypeptide from its NH2-terminus.
