PROTON-TRANSFER IN BENZYL ALCOHOL-DEHYDROGENASE DURING CATALYSIS - ALTERNATE PROTON-RELAY ROUTES

His51 in horse liver alcohol dehydrogenase (ADHE) has been proposed to act as a proton donor/acceptor in the NAD(+)/NADH-dependent oxidation /reduction of alcohol/aldehyde. The residue corresponding to His51 of ADHE is Val51 (Val45 in the protein sequence) in benzyl alcohol dehydr ogenase (BADH) encoded by TOL plasmid pWWO. The 3-D structure of BADH modeled from the crystal structure of ADHE suggests that His47 (His41 in the protein sequence, corresponding to Arg47 in ADHE) of BADH would play the role of His51 in ADHE. To test this hypothesis, mutants of B ADH, in which His47 was replaced by Gln(His47Gln) and/or Val51 was rep laced by His (Val51His), were constructed. The k(cat)/K-m value of the His47Gln mutant for benzyl alcohol was 125-fold lower than that of wi ld-type BADH, while the k(cat)/K-m value of the His47Gln/Val51His doub le mutant was 12-fold higher than that of the His47Gln mutant. The k(c at)/K-m value of the His47Gln mutant increased with increasing concent ration of exogenous amines. These results suggest that His47 in wild-t ype BADH, exogenous amines in the His47Gln mutant, and His51 in the do uble mutant act as a general base catalyst during alcohol oxidation.