Inactivation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid

Abstract

Koningic acid, a sesquiterpene antibiotic, is a specific inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogense ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12). In the presence of 3 mM of NAD +, koningic acid irreversibly inactivated the enzyme in a time-dependent manner. The pseudo-first-order rate constant for inactivation ( k app ) was dependent on koningic acid concentration in saturate manner, indicating koningic acid and enzyme formed a reversible complex prior to the formation of an inactive, irreversible complex; the inactivation rate ( k 3 ) was 5.7 · 10 − s −1 , with dissociation constant for inactivation ( K inact ) of 1.6 μM. The inhibition was competitive against glyceraldehyde 3-phosphate with a K i of 1.1 μM, where the K m for glyceraldehyde 3-phosphate was 90 μM. Koningic acid inhibition was uncompetitive with respect to NAD +. The presence of NAD + accelerated the inactivation. In its absence, the charcoal-treated NAD +-free enzyme showed a 220-fold decreas in apparent rate constant for inactivation, indicating that koningic acid sequentially binds to the enzyme next to NAD +. The enzyme, a tetramer, was inactivated when maximum two sulfhydryl groups, possibly cysteine residues at the active sites of the enzyme, were modified by the binding of koningic acid. These observations demonstrate that koningic acid is an active-site-directed inhibitor which reacts predominantly with the NAD +-enzyme complex.