Catalytic reaction profile for NADH-dependent reduction of aromatic aldehydes by xylose reductase from Candida tenuis.
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 (k(cat) and K(m)) 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 k(cat) and k(cat)/K(m) for enzymic reactions with meta-substituted benzaldehydes have been obtained by using NADH (2)H-labelled in the pro-R C4-H position, and equilibrium constants for the conversion of these aldehydes into the corresponding alcohols (K(eq)) have been measured in the presence of NAD(H) and enzyme. Aldehyde dissociation constants (K(d)) and the hydride transfer rate constant (k(7)) have been calculated from steady-state rate and KIE data. Quantitative structure-activity relationship analysis was used to factor the observed substituent dependence of k(cat)/K(m) into a major electronic effect and a productive positional effect of the para substituent. k(cat)/K(m) (after correction for substituent position) and K(eq) obeyed log-linear correlations over the substituent parameter, Hammett sigma, giving identical slope values (rho) of +1.4 to +1.7, whereas the same Hammett plot for logK(d) yielded rho=-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 k(cat) (1.8) and k(cat)/K(m) (2.7), and likewise k(7), 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 rho 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).
Mayr
Peter
P
Division of Biochemical Engineering, Institute of Food Technology, University of Agricultural Sciences Vienna (BOKU), Muthgasse 18, Austria.
Nidetzky
Bernd
B
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000438:Alcohols / Q000737:chemistry; D000449:Aldehyde Reductase / Q000737:chemistry*; D000447:Aldehydes / Q000737:chemistry*; D000074408:Aldo-Keto Reductases; D001665:Binding Sites; D001669:Biochemical Phenomena; D001671:Biochemistry; D002175:Candida / Q000201:enzymology*; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016014:Linear Models; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D011522:Protons; D021281:Quantitative Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors; D014994:Xylose / Q000737:chemistry
D000438:Alcohols; D000447:Aldehydes; D011522:Protons; D009243:NAD; D014994:Xylose; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase