Proton transfer in benzyl alcohol dehydrogenase during catalysis: Alternate proton-relay routes

被引:26
作者
Inoue, J
Tomioka, N
Itai, A
Harayama, S
机构
[1] Marine Biotechnol Inst, Kamaishi, Iwate 026, Japan
[2] Inst Med Mol Design, Bunkyo Ku, Tokyo 113, Japan
关键词
D O I
10.1021/bi970726g
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 dehydrogenase (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 BADH, in which His47 was replaced by Gln(His47Gln) and/or Val51 was replaced 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 wild-type BADH, while the k(cat)/K-m value of the His47Gln/Val51His double mutant was 12-fold higher than that of the His47Gln mutant. The k(cat)/K-m value of the His47Gln mutant increased with increasing concentration of exogenous amines. These results suggest that His47 in wild-type BADH, exogenous amines in the His47Gln mutant, and His51 in the double mutant act as a general base catalyst during alcohol oxidation.
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页码:3305 / 3311
页数:7
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