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this analog interacts with the catalytic aspartate carboxyls in the same manner as statine or
hydroxyethylene moieties; whereas, the second hydroxyl forms a hydrogen bond with the NH group of
Gly 76, thereby mimicing the carbonyl oxygen at P 1' of other analogs (see Figure 2).
Phosphorus-Containing Analogs
Aspartic proteinase inhibitors in which the scissile bond is replaced by a phosphinic acid group (shown
below) have been reported [28].
These may mimic the tetrahedral intermediate more closely than statine or hydroxyethylene analogs.
One of the oxygens binds to the carboxyl diad and the other resides adjacent to Tyr75 (pepsin
numbering) forming a hydrogen bond with the outer oxygen of Asp32 [27]. This isostere is very
effective against pepsin. However, it ionizes at physiological pH and the resulting anion is ineffective as
an inhibitor of renin [29].
Fluoroketone Analogs and Implications for Catalysis
Fluoroketone analogs (-CO-CF 2-) have been reported [16, 30] and found to be substantially more potent
than the unhalogenated statone molecules, presumably due to the ease of hydration and greater
complementarity of the resulting hydrated gem-diol with the catalytic site. The structure of a
difluorostatone inhibitor complexed with endothiapepsin [31] revealed interactions that indicate how the
catalytic intermediate is stabilized by the enzyme (Figure 3). One hydroxyl of the hydrated fluoroketone
associates tightly with the aspartate diad in the same position as the statine hydroxyl or the native
solvent molecule and the other hydroxyl is positioned such that it hydrogen bonds to the outer carboxyl
oxygen of Asp32. It has been suggested that the tetrahedral intermediate is uncharged, because if the
carboxyl of Asp32 carries a negative charge instead, the latter can be stabilized by a full complement of
hydrogen bonds donated by the gem-diol intermediate and surrounding protein atoms [31]. The current
mechanistic proposals are based on the key suggestion by Suguna et al. [32] that, although transition
state analogs appear to displace the active-site water molecule located between the two catalytic
aspartate carboxyls, the more weakly bound substrate may not. Instead as the substrate binds, the water
may be partly displaced to a position appropriate for nucleophilic attack on the scissile bond carbonyl.
Details of the proposed mechanism are given in Figure 3.
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