title: 4-Aminobutyrate aminotransferase. Conformational changes induced by reduction of pyridoxal 5-phosphate.
abstract: Conformational changes induced in 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) by conversion of pyridoxal-5-P to pyridoxyl-5-P were examined by two independent methods. The reactivity of the SH groups of the reduced enzyme is increased by chemical modification of the cofactor. 1.8 SH per dimer of modified enzyme react with DTNB, whereas 1.2 SH per dimer of the native enzyme react with the attacking reagent under identical experimental conditions. The modified and native forms of the enzyme bind the fluorescent probe ANS, but the number of binding sites for ANS is increased as result of conversion of P-pyridoxal to P-pyridoxyl. After the conformational changes onset by reduction of the cofactor, the modified enzyme binds one molecule of pyridoxal-5-P with a Kd of 0.1 microM to become catalytically competent. The catalytic site of the reduce enzyme was probed with P-pyridoxal analogs. Like resolved 4-aminobutyrate aminotransferase, the reduced species recognize the phosphorothioate analog and regain 40% of the total enzymatic activity. Since the catalytic parameters of reduced and native 4-aminobutyrate aminotransferase are indistinguishable, it is concluded that the additional catalytic site of the reduced enzyme is functionally identical to that of the native enzyme.
mesh_terms: D000612:4-Aminobutyrate Transaminase / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000648:ultrastructure; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008928:Mitochondria / Q000201:enzymology; D011487:Protein Conformation / Q000187:drug effects; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013552:Swine; D013696:Temperature; D013997:Time Factors
chemical_list: D011732:Pyridoxal Phosphate; D004228:Dithionitrobenzoic Acid; D000612:4-Aminobutyrate Transaminase