The role of noncovalent interactions in the catalytic mechanism of aldose reductase from the yeast Candida tenuis was determined by steady-state kinetic analysis of the NADH-dependent reduction of various aldehydes, differing in hydrophobicity and the hydrogen bonding capability with the binary enzyme-NADH complex. In a series of aliphatic aldehydes, substrate hydrophobicity contributes up to 13.7 kJ/mol of binding energy. The aldehyde binding site of aldose reductase appears to be 1.4 times more hydrophobic than n-octanol and can accommodate a linear alkyl chain with at least seven methylene groups (approximately 14 A in length). Binding energy resulting from interactions at positions 3-6 of the aldehyde is distributed between increasing the catalytic constant 2.6-fold and decreasing the apparent dissociation constant 59-fold. Hydrogen bonding interactions of the enzyme nucleotide complex with the C-2(R) hydroxyl group of the aldehyde are crucial to transition state binding and contribute up to 17 kJ/mol of binding energy. A comparison of the kinetic data of yeast aldose reductase, a key enzyme in the metabolism of D-xylose, and human aldose reductase, a presumably perfect detoxification catalyst [Grimshaw, C. E. (1992) Biochemistry 31, 10139], clearly reflects these differences in physiological function.