1MGO

Horse Liver Alcohol Dehydrogenase Phe93Ala Mutant

Summary for 1MGO

• Entry DOI: 10.2210/pdb1mgo/pdb
• Related: 1HLD 1MG0
• Descriptor: Alcohol Dehydrogenase E chain, ZINC ION, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, ... (6 entities in total)
• Functional Keywords: dehydrogenase, alcohol, nicotinamide coenzyme, mutant, substrate binding site, oxidoreductase
• Biological source: Equus caballus (horse)
• Cellular location: Cytoplasm: P00327
• Total number of polymer chains: 2
• Total formula weight: 81657.20

Authors

Rubach, J.K.,Plapp, B.V. (deposition date: 2002-08-15, release date: 2002-11-13, Last modification date: 2024-02-14)

Primary citation

Rubach, J.K.,Plapp, B.V.
Mobility of Fluorobenzyl Alcohols Bound to Liver Alcohol Dehydrogenases as Determined by NMR and X-ray Crystallographic Studies
Biochemistry, 41:15770-15779, 2002

PubMed 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.

PubMed: 12501206
DOI: 10.1021/bi026581h

Experimental method

X-RAY DIFFRACTION (1.2 Å)