Abstract: In order to examine the substrate-binding site of O-acetylserine sulfhydrylase (OASS) from Escherichia coli (E. coli), we mutated Gln-142 and Phe-143, which exist at a β-turn region in the active site. The mutants retained one molecule of pyridoxal 5’-phosphate (PLP) per subunit and PLP was covalently bound in Schiff base linkage, similar to what was observed for the wild type enzyme. Q142A and F143Y OASS inhibited the reaction with O-acetylserine and the subsequent formation of the amino acrylate intermediate. The F143A, S and D mutants were able to form the amino acrylate intermediate, but the rate was significantly slower than that of the wild type enzyme. These results suggest that mutagenesis of Gln-142 and Phe-143 residues in OASS influence catalytic properties, possibly due to modulation of the substrate-binding site.