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 (19)F 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 (19)F 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 (19)F 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.
Rubach
Jon K
JK
Department of Biochemistry, The University of Iowa, Iowa City, IA 52242, USA.
Plapp
Bryce V
BV
D000409:Alanine / Q000235:genetics; D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001592:Benzyl Alcohols / Q000737:chemistry*; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D005461:Fluorine; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010649:Phenylalanine / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics; D005464:Fluorobenzenes; D005466:Fluorocarbons
D000409:Alanine; D000426:Alcohol Dehydrogenase; D001592:Benzyl Alcohols; D005461:Fluorine; D009243:NAD; D010649:Phenylalanine; D011994:Recombinant Proteins; C087170:2,3,4,5,6-pentafluorobenzyl alcohol; D005464:Fluorobenzenes; D005466:Fluorocarbons