Mobility of Fluorobenzyl Alcohols Bound to Liver Alcohol Dehydrogenases as Determined by NMR and X-ray Crystallographic Studies^,

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.

Fluorine–Protein Interactions and 19F NMR Isotropic Chemical Shifts: An Empirical Correlation with Implications for Drug Design

“…All fluorines (with one exception) with d > À160 ppm in the histogram in Figure 5 c (right) originate from these perfluorinated aromatic compounds (i.e. 1mgo, [35] 3dmz, [36] 3b67 [37] ). The quadrupole moment of hexafluorobenzene is 31.7 10 À40 C·m 2 and is essentially equal in magnitude but opposite in sign to that of benzene (À29 10 À40 C·m 2 ).…”

“…The six electronegative fluorine atoms polarize the electronic charge distribution, resulting in a positive electrostatic potential above and below the aromatic ring and a corresponding negative electrostatic potential on the periphery. [38] In the protein-ligand complex structures 1mgo [35] (CF 5 CH 2 OH in complex with liver alcohol dehydrogenase) and 3b67 [37] (pentafluorophenoxy derivative in complex with androgen receptor ligand binding domain), the sulfur approaches the penta-and hexafluorobenzene ring from above or below (with sulfur···aromatic plane distances of 3.1 and 3.4 , respectively). This is different from what is typically observed for interactions between methionine sulfurs and aromatic side chains.…”

Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93

“…1S), which is slower than the rate constant observed for wild-type enzyme of 180 s −1 [31]. Thus, the F93A substitution has significant effects on the catalysis of hydride transfer, as was also observed in the reaction with benzyl alcohol and benzaldehyde [32]. Substrate deuterium isotope effects with ethanol and benzyl alcohol confirm that the hydrogen transfer does not limit catalytic turnover (Table 6S).…”

“…1 in Ref. 32). Thus, it is not clear why the F93A and S48T/F93A substitutions increase the affinity for NADH (by 10-fold, Table 2).…”

“…A structure for the F93A enzyme previously determined at atomic resolution (1MGO.pdb) is also almost identical to that of the wild-type enzyme, but no clear electron density was apparent for the PFB in the active site [32]. This could be due multiple positions (disorder) because the substrate binding site was enlarged, and NMR results show that the mobility of fluorobenzyl alcohols is increased.…”