COMPARATIVE ROLES OF HISTIDINE-51 IN HUMAN BETA(1)BETA(1) AND THREONINE-51 IN PI-PI ALCOHOL DEHYDROGENASES

Histidine at position 51 of the class I beta 1 alcohol dehydrogenase (ADH) functions as a general base by indirectly abstracting a proton from the alcohol substrate through a hydrogen-bonded proton relay system. The human class II pi-ADH was reported to be polymorphic, having either Ser or Thr, but not His at position 51. It is unknown whether Ser or Thr51 have a catalytic role in ethanol oxidation with pi-ADH. Accordingly, we expressed and purified recombinant mutants of pi-ADH with Thr, Ser, and His at position 51. At pH 6.5, values for V-max/K-m for ethanol were 0.30, 0.10, and 0.09 min(-1) mM(-1) for pi 51Thr, pi 51Ser, and pi 51His ADH, respectively. Hence the effects of the substitutions were much less than the 11-fold decrease in Vmax/K-m observed for beta(1)-ADH when a neutral amino acid (Gln) was substituted for His51. Addition of a buffer base (400 mM glycylglycine) had little effect on V-max/K-m of recombinant pi 51Thr or pi 51Ser ADH, while it increased V-max/K-m for ethanol 7-fold for the beta(1)51Gln ADH. We conclude that there is no evidence for Thr51 of pi-ADH participating in a proton relay similar to that seen in beta(1)-ADH and that a base at position 51 may not be a universal requirement for a functional alcohol dehydrogenase with a moderate efficiency for ethanol oxidation at a physiological pH. (C) 1994 Academic Press,Inc.