Binding-equilibrium and kinetic studies of anthocyanidin reductase from Vitis vinifera

Anthocyanidin reductase from Vitis vinifera catalyzes an NADPH-dependent double reduction of anthocyanidins. At pH 7.5 and 30 °C, steady-state kinetics support a hyperbolic and rapid-equilibrium ordered mechanism, with NADPH binding first, K M(cyan) = 2.82 ± 0.66 μM and K i(NADPH) = 111 ± 23 μM. The chromatographic method of Hummel and Dreyer was used for binding-equilibrium studies of NADPH, NADP + and catechin, at pH 7. This confirmed hyperbolic binding of NADPH and NADP + to the free enzyme, with a single binding site each and with dissociation constants K NADPH = 45.9 ± 2 μM and K NADP+ = 83 ± 5 μM. There was no significant binding of catechin. We conclude (i) that the most likely mechanism is sequential ordered Bi Uni Uni Bi, with NADPH binding first and NADP + released last, and (ii) that internal conversion of the first ternary complex, i.e. that associated with the first hydride transfer, is rate-limiting.