Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 4: Hyperbolic activation of rat liver microsomal NADPH cytochrome C reductase by the novel acetylator 7,8-diacetoxy-4-methylcoumarin

The effect of 7,8-diacetoxy-4-methylcoumarin (DAMC) has been studied on hepatic NADPH cytochrome C reductase-an enzyme participating in the microsomal electron transport. The preincubation of liver microsomes with DAMC resulted in a time-dependent activation of NADPH cytochrome C reductase. The catalytic activity of the enzyme enhanced nearly 600% by 25 mu M concentration of DAMC after 10 min of preincubation. The action of DAMC on the reductase resulted in enhanced nu(max) while K-m remained constant. A plot of 1/nu(max) as a function of DAMC concentration resulted in a non-linear, but rectangular hyperbola indicative of hyperbolic activation. DAMC was also proved to be effective in significantly enhancing the activity of NADPH cytochrome C reductase in vivo. 7,8-Dihydroxy-4-methylcoumarin (DHMC), the deacetylated product of DAMC failed to irreversibly activate the enzyme. The activation effect of DAMC upon the enzyme was abolished by p-hydroxymercury benzoate. The role of a transacetylase in transferring the acetyl group of DAMC to the amino acid(s) of the active site of NADPH cytochrome C reductase causing irreversible enzyme activation is enunciated. (C) 1999 Elsevier Science Ltd. All rights reserved.