Studies of the Enzymic Mechanism of Candida tenuis Xylose Reductase (AKR 2B5): X-ray Structure and Catalytic Reaction Profile for the H113A Mutant

Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldoketo reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 Å structure of H113A mutant bound to NADP⁺, and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k_cat and k_cat/ K_{m xylose} show that, relative to the wild type, the hydride transfer rate constant (k₇ ≈ 0.16 s^-1) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k_cat and k _cat/K_{m xylose} for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k_cat/K_{m xylose}) for the wild type and H113A decreased above apparent pK_a values of 8.85 and 7.63, respectively. The ΔpK_a of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k_cat and k_cat/K_{m aldehyde} into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett σ scale, electronic parameter coefficients (ρ) of +0.64 (k_cat) and +0.78 (k _cat/K_{m aldehyde}) were calculated and clearly differ from ρ(k_cat/K_aldehyde) and ρ(k_cat) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nε2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.