Mechanism of inactivation of pyridoxal phosphate-linked aspartate transaminase by gostatin.
The inactivation mechanism of pyridoxal phosphate-linked mitochondrial aspartate transaminase (pig heart) by gostatin (5-amino-2-carboxy-4-oxo-1,4,5,6-tetrahydropyridine-3-acetic acid), a novel amino acid produced by Streptomyces sumanensis, was investigated. Gostatin is a time-dependent inhibitor of the enzyme giving an enzyme half-life of 1.8 min at 3.1 microM (25 degrees C). The kinetic properties of the inhibitor suggest that it is a suicide substrate (mechanism-based inhibitor) of the enzyme, and the observed Ki is 59 microM and Kcat is 0.11S-1 at 25 degrees C. Incubation of the enzyme with a stoichiometric amount of the inhibitor (1 mol of inhibitor/1 mol of enzyme monomer) results in complete inactivation. Spectrophotometric titration and gel filtration experiments indicate the binding of 1 mol of gostatin with 1 mol of enzyme monomer. Gostatin serves as an efficient titrant for the enzyme. Liberation of a compound having inhibitory activity against the apo-form enzyme from the enzyme-inhibitor complex under denaturing conditions suggests irreversible modification of the cofactor.
D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D000818:Animals; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013053:Spectrophotometry; D013552:Swine
D000600:Amino Acids, Dicarboxylic; D011732:Pyridoxal Phosphate; C042220:gostatin; D001219:Aspartate Aminotransferases