Chemical characterization of biodegradative threonine dehydratases from two enteric bacteria

Some chemical properties of the purified biodegredative threonine dehydratases ( l -threonine hydro-lyase (deaminating), EC 4.2.1.16) from Escherichia coli and Salmonella typhimurium are described. The overall amino acid compositions of the two enzymes appear similar with some variations in several amino acid residues. Tryptic peptide maps show that in S. typhimurium four peptides of E. coli origin are missing, whereas six peptides unique to Salmonella protein are present. Carboxymethylation reaction with iodo[14C]acetate to detect half-cystine residues indicates that peptides 21 and S5 in S. typhimurium, but not in E. coli enzyme, are labeled, and the reverse is true for peptide 22; four other peptides of S. typhimurium have more half-cystine residues than their counterparts in E. coli. In addition, the Salmonella enzyme appears to have several disulfide bonds. Despite these differences, the amino acid sequence of the amino termini of the two proteins reveals a highly conserved structure, with only three out of 25 residues being different. Reduction with tritium-labeled borohydride followed by tryptic fingerprinting of the two proteins shows that one peptide contains active-site pyridoxal phosphate. Modifier binding studies with the S. typhimurium enzyme indicate that pyruvate and glyoxylate occupy separate sites on the enzyme molecules. Further, there are two distinct sites for glyoxylate binding: in the monoglyoxylated form of the enzyme, only peptide 22 becomes labeled, whereas both peptides 22 and 21 of the tetraglyoxylated form of the dehydratase contain bound glyoxylate. These results support the earlier findings that these two metabolites regulate enzyme activity by two separate, mutually exclusive, mechanisms.