2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme

Abstract

Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the l-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme ( M r = 84 000) that requires pyridoxal 5′-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH 4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of ε- N-pyridoxyllysine. Quantitative determinations established tha t 2 mol of pyridoxal 5′-phosphate are bound per mol of dimeric enzyme. After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Ly∗s-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly-Tyr-Thr-Ala-Ala-Arg (where ∗ = the lysine residue in azomethine linkage with pyridoxal 5′-phosphate). This peptide corresponds to residues 235–257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver.