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Mobility of Fluorobenzyl Alcohols Bound to Liver Alcohol Dehydrogenases as Determined by NMR and X-ray Crystallographic Studies

The relationship between substrate mobility and catalysis was studied with wild-type and Phe93Ala (F93A) horse liver alcohol dehydrogenase (ADH). Wild-type ADH binds 2,3,4,5,6-pentafluorobenzyl alcohol in one position as shown by X-ray results, and 19F NMR shows five resonances for the fluorines of...

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Bibliographic Details
Published in:Biochemistry (Easton) 2002-12, Vol.41 (52), p.15770-15779
Main Authors: Rubach, Jon K, Plapp, Bryce V
Format: Article
Language:English
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Summary:The relationship between substrate mobility and catalysis was studied with wild-type and Phe93Ala (F93A) horse liver alcohol dehydrogenase (ADH). Wild-type ADH binds 2,3,4,5,6-pentafluorobenzyl alcohol in one position as shown by X-ray results, and 19F NMR shows five resonances for the fluorines of the bound alcohol. The two meta-fluorines exchange positions with a rate constant of about 4 s-1, indicating that mobility (ring flipping) of the benzyl alcohol is relatively restricted. The wild-type enzyme binds 2,3-difluorobenzyl alcohol in two alternative conformations that are related by a ring flip and a small translation of the fluorinated benzene ring, and the 19F NMR spectrum shows three resonances for the two bound fluorines, consistent with the two orientations. Phe-93 interacts with the bound benzyl alcohols, and the F93A substitution decreases the rate constants for hydride transfer for benzyl alcohol oxidation and benzaldehyde reduction by 7.4- and 130-fold, respectively. The structure of F93A ADH crystallized with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol is similar to the structure of the wild-type enzyme complex except that the pentafluorobenzyl alcohol is not found in one position. The 19F NMR spectrum of the F93A ADH−NAD+−pentafluorobenzyl alcohol complex shows three resonances for the bound fluorines. Line shape analysis of the spectrum suggests the bound pentafluorobenzyl ring undergoes rapid ring-flipping at about 20 000 s-1. The F93A substitution greatly increases the mobility of the benzyl alcohol but modestly and differentially decreases the probability that the substrate is preorganized for hydride transfer.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi026581h