1MGO: Horse Liver Alcohol Dehydrogenase Phe93ala Mutant

Citation:
Abstract
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.
PDB ID: 1MGODownload
MMDB ID: 21232
PDB Deposition Date: 2002/8/15
Updated in MMDB: 2002/12
Experimental Method:
x-ray diffraction
Resolution: 1.2  Å
Source Organism:
Similar Structures:
Biological Unit for 1MGO: dimeric; determined by author and by software (PISA)
Molecular Components in 1MGO
Label Count Molecule
Proteins (2 molecules)
2
Alcohol Dehydrogenase E Chain(Gene symbol: LOC100034242)
Molecule annotation
Chemicals (9 molecules)
1
4
2
2
3
2
4
1
* Click molecule labels to explore molecular sequence information.

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