SUBSTITUTION OF PYRIDOXAL 5'-PHOSPHATE IN THE O-ACETYLSERINE SULFHYDRYLASE FROM SALMONELLA-TYPHIMURIUM BY COFACTOR ANALOGS PROVIDES A TEST OF THE MECHANISM PROPOSED FOR FORMATION OF THE ALPHA-AMINOACRYLATE INTERMEDIATE

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)- dependent enzyme that catalyzes the final step in the de novo synthesi s of L-cysteine in Salmonella typhimurium. Complementary cofactor muta genesis in which the active site PLP is substituted with cofactor anal ogs is used to test the mechanism proposed for the OASS, Data obtained with the pyridoxal 5'-deoxymethylenephosphonate-substituted enzyme su ggest that the binding of OAS as it forms the external Schiff base is such that the acetate side chain is properly positioned for eliminatio n (orthogonal to the developing alpha,beta-double bond) only about 1% of the time, Data support the assignment of an enzyme group with a pK of 6.7 that interacts with the acetyl side chain, maintaining it ortho gonal to the developing alpha,beta-double bond, Similar studies of the 2'-methylpyridoxal 5'-phosphate-substituted enzyme suggest that, alth ough the mechanism is identical to that catalyzed by native OASS, the reaction coordinate for alpha-proton abstraction may be decreased comp ared with that observed for the native enzyme.