The kinetic investigation of D-hydroxyisovalerate dehydrogenase from Fusarium sambucinum

초록

The steady-state investigation of the mechanism of D-hydroxyisovalerate dehydrogenase was performed in order to understand this type of kinetic patterns. The initial velocity was measured with various amounts of both substrates, NADPH and 2-ketoisovalerate. Double reciprocal plots gave patterns that conversed on or near the abscissa, Binding studies indicated that NADPH bound first to the enzyme. The product NADP was found to be a competitive inhibitor with respect to NADPH at a constant concentration of 2-ketoisovalerate, However, it showed noncompetitive inhibition against 2-ketoisovalerate at a fixed amount of NADPH, Another product, D-hydroxyisovalerate, was a non-competitive inhibitor versus NADPH and 2-ketoisovalerate at constant levels of 2-ketoisovalerate and NADPH, respectively. These results were comparable with an ordered bi-bi mechanism, in which NADPH bound first to the enzyme, followed by the binding of 2-ketoisovalerate, NADP(+) is the last product to be released. The ordered reaction manner of D-hydroxyisovalerate dehydrogenase from 2-ketoisovalerate to D-hydroxyisovalerate allows the accurate regulation of valine metabolism and it may lead to the regulation of total biosynthesis of enniatins in the Fusarium species.

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