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  • Title: Binding energy and specificity in the catalytic mechanism of yeast aldose reductases.
    Author: Nidetzky B, Mayr P, Hadwiger P, Stütz AE.
    Journal: Biochem J; 1999 Nov 15; 344 Pt 1(Pt 1):101-7. PubMed ID: 10548539.
    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.
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