CYSTEINE-42 IS IMPORTANT FOR MAINTAINING AN INTEGRAL ACTIVE-SITE FOR O-ACETYLSERINE SULFHYDRYLASE RESULTING IN THE STABILIZATION OF THE ALPHA-AMINOACRYLATE INTERMEDIATE

O-Acetylserine sulfhydrylase-A (OASS-A) is a pyridoxal 5'-phosphate (P LP) dependent enzyme from Salmonella typhimurium that catalyzes the P- 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 wh ich 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 Schi ff 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) B iochemistry 35, 13485-13493], and the sole cysteine in the primary str ucture 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-di rected mutagenesis. The mutant proteins are structurally nearly identi cal to the wild-type enzyme on the basis of W-visible, fluorescence, f ar-UV and cofactor-induced CD, and P-31 NMR studies, but subtle struct ural differences are noted. Kinetic properties of both mutant proteins differ significantly from those of the wild-type enzyme. The C42S mut ant 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 wi ldtype enzyme, while decreases of >200-fold in the OAS:acetate lyase a ctivity and a 30-fold decrease in V for the cysteine synthesis are fou nd for the C42A mutant enzyme. In both cases, however, the pH dependen ce of kinetic parameters for cysteine synthesis and OAS:acetate lyase activity yield, within error, identical pK values. In the three-dimens ional structure of OASS-A, cysteine 42 is located behind the cofactor, pointing away from the active site, toward the interior of the protei n. The dramatic change in the OAS:acetate lyase activity of OASS-A in the C42S and C42A mutant proteins likely results from a localized move ment of the serine hydroxyl (compared to the cysteine thiol) toward ad ditional hydrophilic, hydrogen-bonding groups in C42S, or away from hy drophilic groups for C42A, repositioning structure around and includin g K41. Subtle movement of the epsilon-amino group of K41 may change th e geometry for nucleophilic displacement of the amino acid from PLP, l eading to changes in overall activity and stability of the alpha-amino acrylate intermediate. Data indicate that single amino acid substituti ons that yield only subtle changes in structure can produce large diff erences in reaction rates and overall mechanism.