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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.
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