title: 4-Aminobutyrate aminotransferase: identification of lysine residues connected with catalytic activity.
abstract: Bis-PLP (P'P2-bis[5'-pyridoxal]diphosphate) was used as a probe of the catalytic site of 4-aminobutyrate aminotransferase. It reacts with lysine residues connected with aminotransferase activity and the binding of 1 mol of reduced bis-PLP/enzyme monomer abrogates catalytic activity. The reactive lysine residues are characterized by low pK values (pK = 7.3). The presence of substrate 2-oxoglutarate (4 mM) prevents inactivation of the aminotransferase treated with bis-PLP. After tryptic digestion of the enzyme modified with bis-PLP and reduced with tritiated NaBH4, a radioactive peptide absorbing at 320 nm was separated by reverse-phase high-performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysine residue of monomeric 4-aminobutyrate aminotransferase has reacted with bis-PLP. The sequence of the modified peptide differs from the sequence of the peptide bearing the cofactor pyridoxal-5-P covalently attached to a lysine residue. It is postulated that the modified lysine residue is involved in direct interactions with negatively charged carboxylic groups of 2-oxoglutarate.
authors.affiliation: Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
authors.forename: Y T
authors.initials: YT
authors.lastname: Kim
authors.forename: J E
authors.initials: JE
authors.lastname: Churchich
mesh_terms: D000612:4-Aminobutyrate Transaminase / Q000737:chemistry*; D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D010084:Oxidation-Reduction; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000737:chemistry; D013057:Spectrum Analysis; D013552:Swine; D014357:Trypsin
chemical_list: D011732:Pyridoxal Phosphate; C020797:pyridoxal diphosphate; D000612:4-Aminobutyrate Transaminase; D014357:Trypsin; D008239:Lysine