Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases. |
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| Authors: | B Veerapandian J B Cooper A Sali T L Blundell R L Rosati B W Dominy D B Damon and D J Hoover |
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| Affiliation: | Department of Crystallography, Birkbeck College, London, UK. |
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| Abstract: | We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis. |
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| Keywords: | aspartic proteinase mechanism renin inhibitor tetrahedral intermediate X-ray analysis |
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