The cysteine 43, potentially important in the activity of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium, has been changed to serine. This mutant enzyme (C43S) has been studied in order to gain insight into the structural basis for the binding of inhibitor, substrate and product. UV-visible spectra of C43S exhibit the same spectral change in the presence of OAS as that observed with wild type enzyme, indicating C43S will form an ${alpha}$-aminoacrylate Schiff base intermediate. At pH 6.5, however, the deacetylase activity of C43S is much higher than wild type enzyme indicating that cysteine 43 plays a role in stabilizing the ${alpha}$-aminoacrylate intermediate. The fluoroscence spectrum of C43S exhibits a ratio of emission at 340 to 502 nm of 16.9, reflecting the lower fluorescence of PLP and indicating that the orientation of cofactor and tryptophan are different from that of the wild type enzyme. The emission spectrum of C43S in the presence of OAS gives two maxima at 340 and 535 nm. The 535 nm emission is attributed to the fluoroscence of the ${alpha}$-aminoacrylate intermediate. The visible circular dichroic spectrum was similar to wild type enzyme, but the negative effect observed at 530~550 nm and the molar ellipicity values for the mutant are decreased by about 50% compared to wild type enzyme. The circular dichroic and fluoroscence studies suggest binding of the cofactor is less asymmetric in C43S than in the wild type enzyme.