title: Binding-equilibrium and kinetic studies of anthocyanidin reductase from Vitis vinifera.
abstract: 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.
authors.affiliation: Chimie et Biologie des Membranes et des Nanoobjets UMR CNRS 5248, Bâtiment B8, Avenue des Facultés, Université Bordeaux 1, Talence Cedex, France.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Gargouri
authors.forename: Bernard
authors.initials: B
authors.lastname: Gallois
authors.forename: Jean
authors.initials: J
authors.lastname: Chaudière
mesh_terms: D000872:Anthocyanins / Q000378:metabolism; D002392:Catechin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D013053:Spectrophotometry; D027843:Vitis / Q000201:enzymology*
chemical_list: D000872:Anthocyanins; D008024:Ligands; D009249:NADP; C017154:cyanidin; D002392:Catechin; D009247:NADH, NADPH Oxidoreductases; D010100:Oxygen