1r38
From Proteopedia
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Crystal structure of H114A mutant of Candida tenuis xylose reductase
Overview
Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member, of the aldo-keto 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 A 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 (k(7) approximately 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 DeltapK(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 sigma scale, electronic parameter, coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were, calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(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 Nepsilon2 to, aldehyde O1, assists in catalysis by optimizing the C=O bond charge, separation and orbital alignment in the ternary complex.
About this Structure
1R38 is a Single protein structure of sequence from Candida tenuis with NAP as ligand. Active as Aldehyde reductase, with EC number 1.1.1.21 Full crystallographic information is available from OCA.
Reference
Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant., Kratzer R, Kavanagh KL, Wilson DK, Nidetzky B, Biochemistry. 2004 May 4;43(17):4944-54. PMID:15109252
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