Binding energy and specificity in the catalytic mechanism of yeast aldose reductases

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 synthes ized and used as substrates of the NAD(P)H-dependent aldehyde reduction cat alysed by aldose reductases isolated from the yeasts Candida tenuis, C. int ermedia and Cryptococcus flavus. Steady-state kinetic analysis showed that, in comparison with the parent aldoses, the derivatives were reduced with u p to 3000-fold increased catalytic efficiencies (k(cat)/K-m), reflecting ap parent substrate binding constants (K-m) decreased to as little as 1/250 an d, for D-glucose derivatives, up to 5.5-fold increased maximum initial rate s (k(cat)). The effects on K-m mirror the relative proportion of free aldeh yde 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 th e pyranose ring of sugars; this occurs preferentially with the NADPH-depend ent enzymes. No transition-state stabilization energy seems to be derived f rom 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 specific ity (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 destabil izes the transition state of reduction of the corresponding hydrogen-substi tuted aldoses by approx. 4-9 kJ/mol. The total transition state stabilizati on energy derived from hydrogen bonds between hydroxy groups of the substra te and enzyme-NAD(P)H is similar for all yeast aldose reductases (yALRs), a t 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 hydroge n-bonding, interactions with the substrate for efficient turnover.