A CHANGE IN THE INTERNAL ALDIMINE LYSINE (K-42) IN O-ACETYLSERINE SULFHYDRYLASE TO ALANINE INDICATES ITS IMPORTANCE IN TRANSIMINATION AND AS A GENERAL BASE CATALYST

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate depend ent enzyme that catalyzes a beta-replacement reaction forming L-cystei ne and acetate from O-acetyl-L-serine (GAS) and sulfide. The pyridoxal 5'-phosphate (PLP) is bound at the active site in Schiff base linkage with a lysine. In the present study, the Schiff base lysine was ident ified as lysine 42, and its role in the OASS reaction was determined b y changing it to alanine using site-directed mutagenesis. K42A-OASS is isolated as an external aldimine with methionine or leucine and shows no reaction with the natural substrates. Apo-K42A-OASS can be reconst ituted with PLP, suggesting that K42, is not necessary for cofactor bi nding and formation of the external Schiff base. The apo-K42A-OASS, re constituted with PLP, shows slow formation of the external aldimine bu t does not form the alpha-aminoacrylate intermediate on addition of GA S, suggesting that K42 is involved in the abstraction of the alpha-pro ton in the beta-elimination reaction. The external aldimines formed up on addition of L-Ala or L-Ser are stable and represent a tautomer that absorbs maximally at 420 nm, while L-Cys gives a tautomeric form of t he external aldimine that absorbs at 330 nm, and is also seen in the o verall reaction after addition of primary amines to the assay system. The use of a small primary amine such as ethylamine or bromoethylamine in the assay system leads to the initial formation of an internal (ga mma-thialysine) or external (ethylamine) aldimine followed by the slow formation of the alpha-aminoacrylate intermediate on addition of GAS. Activity could not be fully recovered, and only a single turnover is observed. Data suggest a significant rate enhancement resulting from t he presence of K42 for transimination and general base catalysis.