X-ray Structure of Human Class IV ςς Alcohol Dehydrogenase

The structural determinants of substrate recognition in the human class IV, or ςς, alcohol dehydrogenase (ADH) isoenzyme were examined through x-ray crystallography and site-directed mutagenesis. The crystal structure of ςς ADH complexed with NAD⁺ and acetate was solved to 3-Å resolution. The human β₁β₁ and ςς ADH isoenzymes share 69% sequence identity and exhibit dramatically different kinetic properties. Differences in the amino acids at positions 57, 116, 141, 309, and 317 create a different topology within the ςς substrate-binding pocket, relative to the β₁β₁ isoenzyme. The nicotinamide ring of the NAD(H) molecule, in the ςς structure, appears to be twisted relative to its position in the β₁β₁isoenzyme. In conjunction with movements of Thr-48 and Phe-93, this twist widens the substrate pocket in the vicinity of the catalytic zinc and may contribute to this isoenzyme's high K _m for small substrates. The presence of Met-57, Met-141, and Phe-309 narrow the middle region of the ςς substrate pocket and may explain the substantially decreased K _m values with increased chain length of substrates in ςς ADH. The kinetic properties of a mutant ςς enzyme (ς309L317A) suggest that widening the middle region of the substrate pocket increases K _m by weakening the interactions between the enzyme and smaller substrates while not affecting the binding of longer alcohols, such as hexanol and retinol.