NMR Study of the Inhibition of Pepsin by Glyoxal Inhibitors:  Mechanism of Tetrahedral Intermediate Stabilization by the Aspartyl Proteases

Abstract

Z-Ala-Ala-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of pepsin with a Ki = 89 +/- 24 nM at pH 2.0 and 25 degrees C. Both the ketone carbon (R13COCHO) and the aldehyde carbon (RCO13CHO) of the glyoxal group of Z-Ala-Ala-Phe-glyoxal have been 13C-enriched. Using 13C NMR, it has been shown that when the inhibitor is bound to pepsin, the glyoxal keto and aldehyde carbons give signals at 98.8 and 90.9 ppm, respectively. This demonstrates that pepsin binds and preferentially stabilizes the fully hydrated form of the glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal. From 13C NMR pH studies with glyoxal inhibitor, we obtain no evidence for its hemiketal or hemiacetal hydroxyl groups ionizing to give oxyanions. We conclude that if an oxyanion is formed its pKa must be >8.0. Using 1H NMR, we observe four hydrogen bonds in free pepsin and in pepsin/Z-Ala-Ala-Phe-glyoxal complexes. In the pepsin/pepstatin complex an additional hydrogen bond is formed. We examine the effect of pH on hydrogen bond formation, but we do not find any evidence for low-barrier hydrogen bond formation in the inhibitor complexes. We conclude that the primary role of hydrogen bonding to catalytic tetrahedral intermediates in the aspartyl proteases is to correctly orientate the tetrahedral intermediate for catalysis.

pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor

“…However, if the glyoxal group was coordinated to the zinc atom we would expect the glyoxal hemiacetal hydroxyl groups to also be ionised with the hemiacetal carbon having a chemical shift of ~97 ppm which is not observed. We have also shown that when a glyoxal inhibitor binds to pepsin there is an increase in chemical shift of the glyoxal hemiacetal carbon from 89.8 to 90.9 even though both the hemiacetal and hemiketal hydroxyl groups are not ionised in the free and bound glyoxal [9]. The increase in chemical shift of the glyoxal hemiacetal carbon from 90.4 to 92.0 ppm on binding the UK-370106-CO 13 CHO inhibitor to SCD is similar in magnitude and can be attributed to the difference in the environment of the free and bound species.…”

“…However, it is surprising that with the aspartyl protease pepsin [9] and with the metalloprotease SCD the association rate constant of glyoxal inhibitors is also at least a 50 x slower than the diffusion controlled rate constant. Hydration and dehydration of carbonyl groups is a slow process [39].…”

“…Specific peptide-derived glyoxal inhibitors have been shown to be potent inhibitors of the serine proteases [1][2][3][4][5], the cysteine proteases [5][6][7][8] and the aspartyl proteases [9,10]. Glyoxal inhibitors are most effective with the cysteine proteases and their active site thiol group has been shown to form a thiohemiacetal with the glyoxal aldehyde group [6].…”

“…Glyoxal inhibitors are most effective with the cysteine proteases and their active site thiol group has been shown to form a thiohemiacetal with the glyoxal aldehyde group [6]. The aspartyl proteases bind the fully hydrated glyoxal, which mimics the tetrahedral intermediate thought to be formed during catalysis [9]. In the serine proteases the active site serine hydroxyl group has been shown to form a hemiketal with the keto-carbon of the glyoxal group, this hemiketal mimics the tetrahedral intermediate formed during catalysis [1,2,11].…”

Hemiacetal stabilization in a chymotrypsin inhibitor complex and the reactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin

“…Z-Ala-Ala-Phe-H (Scheme 1A) was synthesized as described by Petrillo et al [4]. Z-Ala-Ala-Phe-13 COOCH 3 and Z-Ala-Ala-Phe-13 COCHO (Scheme 1D) were synthesized as described by Cosgrove et al, [18].…”