Kinetic substituent effects have been used to examine the catalytic reaction profile of xylose reductase from the yeast Candida tenuis, a representative aldo/keto reductase of primary carbohydrate metabolism. Michaelis—Menten parameters (kcat and Km) for NADH-dependent enzymic aldehyde reductions have been determined using a homologous series of benzaldehyde derivatives in which substituents in meta and para positions were employed to systematically perturb the properties of the reactive carbonyl group. Kinetic isotope effects (KIEs) on kcat and kcat/Km for enzymic reactions with meta-substituted benzaldehydes have been obtained by using NADH 2H-labelled in the pro-R C4-H position, and equilibrium constants for the conversion of these aldehydes into the corresponding alcohols (Keq) have been measured in the presence of NAD(H) and enzyme. Aldehyde dissociation constants (Kd) and the hydride transfer rate constant (k7) have been calculated from steady-state rate and KIE data. Quantitative structure—activity relationship analysis was used to factor the observed substituent dependence of kcat/Km into a major electronic effect and a productive positional effect of the para substituent. kcat/Km (after correction for substituent position) and Keq obeyed log-linear correlations over the substituent parameter, Hammett sigma, giving identical slope values (ρ) of +1.4 to +1.7, whereas the same Hammett plot for logKd yielded ρ =-1.5. This leads to the conclusion that electron-withdrawing substituents facilitate the reaction and increase binding to about the same extent. KIE values for kcat (1.8) and kcat/Km (2.7), and likewise k7, showed no substituent dependence. Therefore, irrespective of the observed changes in reactivity over the substrate series studied no shift in the character of the rate-limiting transition state of hydride transfer occurred. The signs and magnitudes of ρ values suggest this transition state to be product-like in terms of charge development at the reactive carbon. Structure—reactivity correlations reveal active-site homologies among NADPH-specific and dual NADPH/NADH-specific yeast xylose reductases and across two aldo/keto reductase families in spite of the phylogenetic separation of the host organisms producing xylose reductase (family 2B) and aldehyde reductase (family 1A).
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September 2002
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Research Article|
September 15 2002
Catalytic reaction profile for NADH-dependent reduction of aromatic aldehydes by xylose reductase from Candida tenuis
Peter MAYR;
Peter MAYR
∗Division of Biochemical Engineering, Institute of Food Technology, University of Agricultural Sciences Vienna (BOKU), Muthgasse 18, A-1190 Vienna, Austria,
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Bernd NIDETZKY
Bernd NIDETZKY
1
∗Division of Biochemical Engineering, Institute of Food Technology, University of Agricultural Sciences Vienna (BOKU), Muthgasse 18, A-1190 Vienna, Austria,
†Institute of Biotechnology, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria
1To whom correspondence should be addressed, at the Institute of Biotechnology, Graz University of Technology (e-mail bernd.nidetzky@tugraz.at).
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Publisher: Portland Press Ltd
Received:
January 11 2002
Revision Received:
April 30 2002
Accepted:
May 10 2002
Accepted Manuscript online:
May 10 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (3): 889–899.
Article history
Received:
January 11 2002
Revision Received:
April 30 2002
Accepted:
May 10 2002
Accepted Manuscript online:
May 10 2002
Citation
Peter MAYR, Bernd NIDETZKY; Catalytic reaction profile for NADH-dependent reduction of aromatic aldehydes by xylose reductase from Candida tenuis. Biochem J 15 September 2002; 366 (3): 889–899. doi: https://doi.org/10.1042/bj20020080
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