title: 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 (Mr = 84,000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of epsilon-N-pyridoxyllysine. Quantitative determinations established that 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-Lys*-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.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor.
authors.forename: J J
authors.initials: JJ
authors.lastname: Mukherjee
authors.forename: E E
authors.initials: EE
authors.lastname: Dekker
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D014357:Trypsin
chemical_list: D000596:Amino Acids; D011732:Pyridoxal Phosphate; D000123:Acetyltransferases; C020679:glycine acetyltransferase; D014357:Trypsin; D008239:Lysine