NONCOVALENT ENZYME-SUBSTRATE INTERACTIONS IN THE CATALYTIC MECHANISM OF YEAST ALDOSE REDUCTASE

The role of noncovalent interactions in the catalytic mechanism of ald ose reductase from the yeast Candida tenuis was determined by steady-s tate kinetic analysis of the NADH-dependent reduction of various aldeh ydes, differing in hydrophobicity and the hydrogen bonding capability with the binary enzyme NADH complex. In a series of aliphatic aldehyde s, substrate hydrophobicity contributes up to 13.7 kJ/mol of binding e nergy. The aldehyde binding site of aldose reductase appears to be 1.4 times more hydrophobic than n-octanol and can accommodate a linear al kyl chain with at least seven methylene groups (approximate to 14 Angs trom in length). Binding energy resulting from interactions at positio ns 3-6 of the aldehyde is distributed between increasing the catalytic constant 2.6-fold and decreasing the apparent dissociation constant 5 9-fold Hydrogen bonding interactions of the enzyme nucleotide complex with the C-2(R) hydroxyl group of the aldehyde are crucial to transiti on slate binding and contribute up to 17 kJ/mol of binding energy. A c omparison of the kinetic data of yeast aldose reductase, a key enzyme in the metabolism of D-xylose, and human aldose reductase, a presumabl y perfect detoxification catalyst [Grimshaw, C. E.(1992) Biochemistry 31, 10139], clearly reflects these differences in physiological functi on.