1r38
From Proteopedia
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'''Crystal structure of H114A mutant of Candida tenuis xylose reductase''' | '''Crystal structure of H114A mutant of Candida tenuis xylose reductase''' | ||
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[[Category: Nidetzky, B.]] | [[Category: Nidetzky, B.]] | ||
[[Category: Wilson, D K.]] | [[Category: Wilson, D K.]] | ||
| - | [[Category: | + | [[Category: Aldo-keto reductase]] |
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| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 07:01:20 2008'' | |
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Revision as of 04:01, 3 May 2008
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. 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 Page seeded by OCA on Sat May 3 07:01:20 2008
