X-ray Structure of Human Class IV ςς Alcohol Dehydrogenase

• Published in: The Journal of biological chemistry 1997-07, Vol.272 (30), p.18558-18563
• Main Authors: Xie, Peiguang, Parsons, Stephen H., Speckhard, David C., Bosron, William F., Hurley, Thomas D.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.30.18558

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 Km 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 Km 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 Km by weakening the interactions between the enzyme and smaller substrates while not affecting the binding of longer alcohols, such as hexanol and retinol.