Hydroxybenzoyl-CoA reductase: coupling kinetics and electrochemistry to derive enzyme mechanisms

Biochemistry
A El KasmiS W Ragsdale

Abstract

Hydroxybenzoyl-CoA reductase (HBCR) is an iron-sulfur protein that is involved in the metabolism of aromatic compounds. It catalyzes the two-electron reduction of hydroxybenzoyl-CoA to benzoyl-CoA. In the work described here, kinetic schemes were derived for HBCR and for several classes of redox enzymes and redox-activated enzymes. Introduction of the Nernst equation into the rate laws led to the development of novel relationships between the ambient redox potential, the midpoint potential of the enzyme active site, and the kinetic parameter, V/K. By coupling electrochemistry and steady-state kinetics, mechanistic information could be obtained that could not be determined by either method alone. For HBCR, the relationship between the kinetic parameter V/K and the ambient electrochemical potential of the assay mixture was found to be: apparent V/Km = Vmax/(Km(1 + exp[nF/RT(E - E(o)e)])), where n is the number of electrons involved in the redox process, F is the Faraday constant, R is the gas constant, T is the temperature in K, E is the applied potential, and E(o)e is the redox potential of a redox-active catalytic site on the enzyme. Coupling kinetics with electrochemistry yielded the E(o)e (-350 mV vs NHE) for HBCR and maximum...Continue Reading

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