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Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant.
Biochemistry. 2004 May 04; 43(17):4944-54.B

Abstract

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.

Authors+Show Affiliations

Institute of Biotechnology, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15109252

Citation

Kratzer, Regina, et al. "Studies of the Enzymic Mechanism of Candida Tenuis Xylose Reductase (AKR 2B5): X-ray Structure and Catalytic Reaction Profile for the H113A Mutant." Biochemistry, vol. 43, no. 17, 2004, pp. 4944-54.
Kratzer R, Kavanagh KL, Wilson DK, et al. Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant. Biochemistry. 2004;43(17):4944-54.
Kratzer, R., Kavanagh, K. L., Wilson, D. K., & Nidetzky, B. (2004). Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant. Biochemistry, 43(17), 4944-54.
Kratzer R, et al. Studies of the Enzymic Mechanism of Candida Tenuis Xylose Reductase (AKR 2B5): X-ray Structure and Catalytic Reaction Profile for the H113A Mutant. Biochemistry. 2004 May 4;43(17):4944-54. PubMed PMID: 15109252.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant. AU - Kratzer,Regina, AU - Kavanagh,Kathryn L, AU - Wilson,David K, AU - Nidetzky,Bernd, PY - 2004/4/28/pubmed PY - 2004/8/13/medline PY - 2004/4/28/entrez SP - 4944 EP - 54 JF - Biochemistry JO - Biochemistry VL - 43 IS - 17 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/15109252/Studies_of_the_enzymic_mechanism_of_Candida_tenuis_xylose_reductase__AKR_2B5_:_X_ray_structure_and_catalytic_reaction_profile_for_the_H113A_mutant_ L2 - https://dx.doi.org/10.1021/bi035833r DB - PRIME DP - Unbound Medicine ER -