A change in the internal aldimine lysine (K42) 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 dependent enzyme that catalyzes a beta-replacement reaction forming L-cysteine and acetate from O-acetyl-L-serine (OAS) 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 identified as lysine 42, and its role in the OASS reaction was determined by 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 reconstituted with PLP, suggesting that K42 is not necessary for cofactor binding and formation of the external Schiff base. The apo-K42A-OASS, reconstituted with PLP, shows slow formation of the external aldimine but does not form the alpha-aminoacrylate intermediate on addition of OAS, suggesting that K42 is involved in the abstraction of the alpha-proton in the beta-elimination reaction. The external aldimines formed upon 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 the external aldimine that absorbs at 330 nm, and is also seen in the overall 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 (gamma-thialysine) or external (ethylamine) aldimine followed by the slow formation of the alpha-aminoacrylate intermediate on addition of OAS. Activity could not be fully recovered, and only a single turnover is observed. Data suggest a significant rate enhancement resulting from the presence of K42 for transimination and general base catalysis.