Transient kinetics and intermediates formed during the electron transfer reaction catalyzed by Candida albicans estrogen binding protein

The transient kinetics of the reaction of the estrogen binding protein (EBP1) from Candida albicans in which hydride is transferred from NADPH to trans-2-hexenal (HXL) in two half-reactions were analyzed using UV-visible spectrophotometric and fluorometric stopped-flow techniques. The simplest model of the first half-reaction involves four steps including very rapid, tight binding (Kd less than or equal to 50 nM) characterized by loss of NADPH fluorescence, subsequent rapid formation of a charge-transfer complex between NADPH and oxidized enzyme-bound flavin mononucleotide (FMN) cofactor, followed by rate-limiting reduction of the FMN, and finally dissociation of the oxidized pyridine nucleotide. The UV-visible absorbance behavior of this half-reaction is described by two apparent phases, with k(obs)(f) = 355 +/- 6 and k(obs)(s) = 3.30 +/- 0.03 s(-1), for the fast and slow phases, respectively. The reaction has also been evaluated in terms of the full, multi-equilibrium reaction scheme, and microscopic rate constants that lead to the observed behavior have been determined through convergent experimental techniques and computer simulation. Significant intrinsic kinetic isotope effects were noted on both the bond cleavage step and the preceding formation of the charge-transfer complex. The enzyme is reoxidized by transfer of hydride from the FMN to HXL in the second half-reaction that appears to consist of substrate binding to form a Michaelis-type complex and the subsequent chemical step. Characterization of the reaction in this simple manner allows determination of an apparent Kd = 100 +/- 9 mu M for the reduced enzyme.HXL complex.