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. 1992 Mar;1(3):322–328. doi: 10.1002/pro.5560010303

Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

B Veerapandian 1, J B Cooper 1, A Sali 1, T L Blundell 1, R L Rosati 1, B W Dominy 1, D B Damon 1, D J Hoover 1
PMCID: PMC2142209  PMID: 1304340

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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Selected References

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