X-RAY STRUCTURE OF HUMAN CLASS-IV SIGMA-SIGMA ALCOHOL-DEHYDROGENASE -STRUCTURAL BASIS FOR SUBSTRATE-SPECIFICITY

The structural determinants of substrate recognition in the human clas s IV, or sigma sigma, alcohol dehydrogenase (ADH) isoenzyme were exami ned through x-ray crystallography and site-directed mutagenesis. The c rystal structure of sigma sigma ADH complexed with NAD(+) and acetate was solved to 3-Angstrom resolution. The human beta(1) beta(1) and sig ma sigma ADH isoenzymes share 69% sequence identity and exhibit dramat ically different kinetic properties. Differences in the amino acids at positions 57, 116, 141, 309, and 317 create a different topology with in the sigma sigma substrate-binding pocket, relative to the beta(1) b eta(1) isoenzyme. The nicotinamide ring of the NAD(H) molecule, in the sigma sigma structure, appears to be twisted relative to its position in the beta(1) beta(1) isoenzyme. In conjunction with movements of Th r-48 and Phe-93, this twist widens the substrate pocket in the vicinit y 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 n arrow the middle region of the sigma sigma substrate pocket and may ex plain the substantially decreased K-m values with increased chain leng th of substrates in sigma sigma ADH. The kinetic properties of a mutan t sigma sigma enzyme (sigma 309L317A) suggest that widening the middle region of the substrate pocket increases K-m by weakening the interac tions between the enzyme and smaller substrates while not affecting th e binding of longer alcohols, such as hexanol and retinol.