Abstract
The inactivation mechanism of pyridoxal phosphate-linked mitochondrial aspartate transaminase (pig heart) by gostatin (5-amino-2-carboxy-4-oxo-1, 4, 5, 6-tetrahydro-pyridine-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 μM (25°C). The kinetic properties of the inhibitor suggest that it is a suicide substrate (mechanism-based inhibitor) of the enzyme, and the observed K1 is 59 μM and Kcat is 0.11 s-1 at 25°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 mot 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.
