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 degrees C, steady-state kinetics support a hyperbolic and rapid-equilibrium ordered mechanism, with NADPH binding first, K(M(cyan))=2.82+/-0.66microM and K(i(NADPH))=111+/-23microM. 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+/-2microM and K(NADP+)=83+/-5microM. 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.