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in the active site of an enzyme or drug-design algorithms such as inhibitor docking may be inappropriate
in some cases if they do not adequately model considerable plasticity in the binding site.
A closer look at the binding of zopolrestat shows that it is dominated by extensive hydrophobic contacts
between the protein and the inhibitor. These include side chains from Trp20, Tyr48, Trp79, Trp111,
Phe115, Phe122, Trp219, Ala299, Leu300, Tyr309, and Pro310 (Figure 7). This is not surprising given
the apolar nature of the enzyme's active site as determined by the holoenzyme structure. What is
surprising is that the inhibitor created the part of its own binding site that the benzothiazole rings occupy
by “burrowing” into the hydrophobic core of the protein to carve out a region with very good steric
complementarity to this moiety. It does this rather than binding in the solvent-exposed hydrophobic
binding groove that is seen in the holoenzyme structure.
The remaining interactions involving hydrogen bonds and salt links also appear to be very important in
inhibitor binding. With the exception of one of the fluorine atoms, all atoms that are able to engage in
hydrogen bonding do so. The carboxylate, which is seen in so many aldose reductase inhibitors, is saltlinked
to His110, which is located very near the catalytic site (Figure 7). Presumably, the carboxylate in
the other inhibitors plays the same role and could be used as an anchor when modeling these into the
active site.
Inhibition studies involving ALR2 have indicated noncompetitive inhibition for virtually all compounds
examined to date when the forward (reduction) reaction is monitored. This mode of inhibition is often
interpreted as meaning that the inhibitor binds to a site on the enzyme that is independent of the catalytic
site. Kinetic and competition studies have both led to this conclusion in the case of ALR2 [24,25]. The
crystal structure of the enzyme complexed with both the NADPH cofactor and zopolrestat, however,
clearly shows the inhibitor occupying the region directly above the nicotinamide of the NADPH and,
therefore, the active site (Figures 5, 6, and 7).
Most previous inhibition studies reported noncompetitive and/or uncompetitive inhibition patterns when
aldose reductase inhibitors were examined in the forward direction, i.e. inhibition of NADPH-dependent
aldehyde reduction. With the finding that the overall rate-limiting step in the direction of aldehyde
reduction is at the level of structural isomerization following alcohol product release [26,27], it is not
surprising that lack of competitive inhibition would be observed in such standard double reciprocal
plots. To further complicate matters, many aldose reductase inhibitors were not recognized in previous
studies as tight-binding inhibitors and were inappropriately evaluated using Michaelis-Menten kinetics.
Thus, noncompetitive or uncompetitive inhibition patterns were previously reported for inhibitors that
were subsequently shown to bind directly at the active site. Recent structure-function and kinetic studies
have revealed important details concerning the structural basis for the catalytic
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