S-adenosylhomocysteinase from rat liver. Amino acid sequences of the peptides containing active site cysteine residues modified by treatment with 5'-p-fluorosulfonylbenzoyladenosine.

Abstract 5'-p-Fluorosulfonylbenzoyladenosine (FSBA) inactivates rat liver S-adenosylhomocysteinase exhibiting characteristics of an active site-directed reagent. The inactivation is not associated with the covalent binding of the reagent, but is correlated with the loss of 2 sulfhydryl groups/enzyme subunit (Takata, Y., and Fujioka, M. (1984) Biochemistry 23, 4357-4362). Treatment of the FSBA-inactivated enzyme with iodoacetate in the absence of reducing agent and then with [14C] iodoacetate after reduction with 2-mercaptoethanol yielded the enzyme containing approximately 2 mol of radiolabeled S-carboxymethylcysteine/mol of subunit. Analysis of tryptic peptides showed that the radioactivity was associated with 2 carboxymethylcysteine-containing peptides whose amino acid sequences were: Trp-Ser-Ser-Cys(Cm)-Asn-Ile-Phe-Ser-Thr-Gln-Asp-His-Ala-Ala-Ala-Ala-Ile- Ala-Lys and Gly-Glu-Thr-Asp-Glu-Glu-Tyr-Leu-Trp-Cys(Cm)-Ile-Glu-Gln-Thr-Leu-His-Phe- Lys, respectively. These results indicate that the inactivation of S-adenosylhomocysteinase by FSBA is the consequence of formation of a disulfide between two specific cysteine residues on each of the four identical subunits.