title: Binding energy and specificity in the catalytic mechanism of yeast aldose reductases.
abstract: Derivatives of d-xylose and d-glucose, in which the hydroxy groups at C-5, and C-5 and C-6 were replaced by fluorine, hydrogen and azide, were synthesized and used as substrates of the NAD(P)H-dependent aldehyde reduction catalysed by aldose reductases isolated from the yeasts Candida tenuis, C. intermedia and Cryptococcus flavus. Steady-state kinetic analysis showed that, in comparison with the parent aldoses, the derivatives were reduced with up to 3000-fold increased catalytic efficiencies (k(cat)/K(m)), reflecting apparent substrate binding constants (K(m)) decreased to as little as 1/250 and, for d-glucose derivatives, up to 5.5-fold increased maximum initial rates (k(cat)). The effects on K(m) mirror the relative proportion of free aldehyde that is available in aqueous solution for binding to the binary complex enzyme-NAD(P)H. The effects on k(cat) reflect non-productive binding of the pyranose ring of sugars; this occurs preferentially with the NADPH-dependent enzymes. No transition-state stabilization energy seems to be derived from hydrogen-bonding interactions between enzyme-NAD(P)H and positions C-5 and C-6 of the aldose. In contrast, unfavourable interactions with the C-6 group are used together with non-productive binding to bring about specificity (6-10 kJ/mol) in a series of d-aldoses and to prevent the reaction with poor substrates such as d-glucose. Azide introduced at C-5 or C-6 destabilizes the transition state of reduction of the corresponding hydrogen-substituted aldoses by approx. 4-9 kJ/mol. The total transition state stabilization energy derived from hydrogen bonds between hydroxy groups of the substrate and enzyme-NAD(P)H is similar for all yeast aldose reductases (yALRs), at approx. 12-17 kJ/mol. Three out of four yALRs manage on only hydrophobic enzyme-substrate interactions to achieve optimal k(cat), whereas the NAD(P)H-dependent enzyme from C. intermedia requires additional, probably hydrogen-bonding, interactions with the substrate for efficient turnover.
authors.affiliation: Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur, Muthgasse 18, A-1190 Vienna, Austria. nide@mail.boku.ac.at
authors.forename: B
authors.initials: B
authors.lastname: Nidetzky
authors.forename: P
authors.initials: P
authors.lastname: Mayr
authors.forename: P
authors.initials: P
authors.lastname: Hadwiger
authors.forename: A E
authors.initials: AE
authors.lastname: Stütz
mesh_terms: D000449:Aldehyde Reductase / Q000378:metabolism*; D002175:Candida / Q000201:enzymology; D002384:Catalysis; D003454:Cryptococcus / Q000201:enzymology; D004734:Energy Metabolism; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014994:Xylose / Q000031:analogs & derivatives / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology*
chemical_list: D009243:NAD; D009249:NADP; D014994:Xylose; D000449:Aldehyde Reductase; D005947:Glucose