Cysteine 42 is important for maintaining an integral active site for O-acetylserine sulfhydrylase resulting in the stabilization of the alpha-aminoacrylate intermediate.

Abstract

O-Acetylserine sulfhydrylase-A (OASS-A) is a pyridoxal 5'-phosphate (PLP) dependent enzyme from Salmonella typhimurium that catalyzes the beta-replacement of acetate in O-acetyl-L-serine (OAS) by sulfide to give L-cysteine. The reaction occurs via a ping-pong kinetic mechanism in which alpha-aminoacrylate in Schiff base with the active site PLP is an intermediate [Cook, P. F., Hara, S., Nalabolu, S. R., and Schnackerz, K. D. (1992) Biochemistry 31, 2298-2303]. The sequence around the Schiff base lysine (K41) has been determined [Rege, V. D., Kredich, N. M., Tai, C.-H., Karsten, W. E., Schnackerz, K. D., & Cook, P. F. (1996) Biochemistry 35, 13485-13493], and the sole cysteine in the primary structure is immediately C-terminal to the lysine. In an effort to assess the role of C42, it has been changed to serine and alanine by site-directed mutagenesis. The mutant proteins are structurally nearly identical to the wild-type enzyme on the basis of UV-visible, fluorescence, far-UV and cofactor-induced CD, and 31P NMR studies, but subtle structural differences are noted. Kinetic properties of both mutant proteins differ significantly from those of the wild-type enzyme. The C42S mutant exhibits a > 50-fold increase in the OAS:acetate lyase activity and a 17-fold decrease in V for the cysteine synthesis compared to the wild-type enzyme, while decreases of > 200-fold in the OAS: acetate lyase activity and a 30-fold decrease in V for the cysteine synthesis are found for the C42A mutant enzyme. In both cases, however, the pH dependence of kinetic parameters for cysteine synthesis and OAS: acetate lyase activity yield, within error, identical pK values. In the three-dimensional structure of OASS-A, cysteine 42 is located behind the cofactor, pointing away from the active site, toward the interior of the protein. The dramatic change in the OAS:acetate lyase activity of OASS-A in the C42S and C42A mutant proteins likely results from a localized movement of the serine hydroxyl (compared to the cysteine thiol) toward additional hydrophilic, hydrogen-bonding groups in C42S, or away from hydrophilic groups for C42A, repositioning structure around and including K41. Subtle movement of the epsilon-amino group of K41 may change the geometry for nucleophilic displacement of the amino acid from PLP, leading to changes in overall activity and stability of the alpha-aminoacrylate intermediate. Data indicate that single amino acid substitutions that yield only subtle changes in structure can produce large differences in reaction rates and overall mechanism.