Structures of horse liver alcohol dehydrogenase complexed with NAD+ and substituted benzyl alcohols

Biochemistry. 1994 May 3;33(17):5230-7. doi: 10.1021/bi00183a028.

Abstract

Structures of the enzyme complexed with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol were determined by X-ray crystallography at a resolution of 2.1 A and to a refinement R value of 18.3% for a monoclinic (P2(1)) form and to 2.4 A and an R value of 18.9% for a triclinic crystal form. The pentafluorobenzyl alcohol does not react, due to electron withdrawal by the fluorine atoms. A structure with NAD+ and p-bromobenzyl alcohol in the monoclinic form was also determined at 2.5 A and an R value of 16.7%. The conformations of the subunits in the monoclinic and triclinic crystal forms are very similar. The dimer is the asymmetric unit, and a rigid body rotation closes the cleft between the coenzyme and catalytic domains upon complex formation. In the monoclinic form, this conformational change is described by a rotation of 9 degrees in one subunit and 10 degrees in the other. The pentafluoro- and p-bromobenzyl alcohols bind in overlapping positions. The hydroxyl group of each alcohol is ligated to the catalytic zinc and participates in an extensive hydrogen-bonded network that includes the imidazole group of His-51, which can act as a base and shuttle a proton to solvent. The hydroxymethyl carbon of the pentafluorobenzyl alcohol is 3.4 A from C4 of the nicotinamide ring, and the pro-R hydrogen is in a good position for direct transfer to C4. The p-bromobenzyl alcohol may react after small rotations around single bonds of the alcohol. These structures should approximate the active Michaelis-Menten complexes.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alcohol Dehydrogenase / chemistry*
  • Alcohol Dehydrogenase / metabolism*
  • Amino Acid Sequence
  • Animals
  • Benzyl Alcohols / metabolism*
  • Binding Sites
  • Crystallography, X-Ray / methods
  • Fourier Analysis
  • Histidine
  • Horses
  • Hydrogen Bonding
  • Liver / enzymology*
  • Macromolecular Substances
  • NAD / metabolism*
  • Protein Conformation*
  • Thermodynamics

Substances

  • Benzyl Alcohols
  • Macromolecular Substances
  • NAD
  • 2,3,4,5,6-pentafluorobenzyl alcohol
  • Histidine
  • Alcohol Dehydrogenase