Structural basis for substrate specificity differences of horse liver alcohol dehydrogenase isozymes

A structure determination in combination with a kinetic study of the steroi d converting isozyme of horse liver alcohol dehydrogenase, SS-ADH, is prese nted. Kinetic parameters for the substrates, 5 beta -androstane-3 beta ,17 beta -ol, 5 beta -androstane-17 beta -ol-3-one, ethanol, and various second ary alcohols and the corresponding ketones are compared for the SS- and EE- isozymes which differ by nine amino acid substitutions and one deletion. Di fferences in substrate specificity and stereoselectivity are explained on t he basis of individual kinetic rate constants for the underlying ordered bi -bi mechanism. SS-ADH was crystallized in complex with 3 alpha ,7 alpha ,12 alpha -trihydroxy-5 beta -cholan-24-acid (cholic acid) and NAD(+), but mic rospectrophotometric analysis of single crystals proved it to be a mixed co mplex containing 60-70% NAD(+) and 30-40% NADH. The crystals belong to the space group P2(1) with cell dimensions a = 55.0 Angstrom, b = 73.2 Angstrom , c = 92.5 Angstrom, and beta = 102.5 degrees. Angstrom 98% complete data s et to 1.54-Angstrom resolution was collected at 100 K using synchrotron rad iation. The structure was solved by the molecular replacement method utiliz ing EE-ADH as the search model. The major structural difference between the isozymes is a widening of the substrate channel. The largest shifts in C-a lpha carbon positions (about 5 Angstrom) are observed in the loop region, i n which a deletion of Asp115 is found in the SS isozyme. SS-ADH easily acco mmodates cholic acid, whereas steroid substrates of similar bulkiness would not fit into the EE-ADH substrate site. In the ternary complex with NAD(+) /NADH, we find that the carboxyl group of cholic acid ligates to the active site zinc ion, which probably contributes to the strong binding in the ter nary NAD(+) complex.