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The X-ray crystal structures of native penicillopepsin and of its complex with a
synthetic analogue of the inhibitor pepstatin have been refined recently at
1.8-A resolution. These highly refined structures permit a detailed examination
of peptide hydrolysis in the aspartic proteinases. Complexes of penicillopepsin
with substrate and catalytic intermediates were modeled, by using computer
graphics, with minimal perturbation of the observed inhibitor complex. A
thallium ion binding experiment shows that the position of solvent molecule O39,
between Asp-33(32) and Asp-213(215) in the native structure, is favorable for
cations, a fact that places constraints on possible mechanisms. A mechanism for
hydrolysis is proposed in which Asp-213(215) acts as an electrophile by
protonating the carbonyl oxygen of the substrate, thereby polarizing the
carbon-oxygen bond, a water molecule bound to Asp-33(32) (O284 in the native
structure) attacks the carbonyl carbon as the nucleophile in a general-base
mechanism, the newly pyramidal peptide nitrogen is protonated, either from the
solvent after nitrogen inversion or by an internal proton transfer via
Asp-213(215) from a hydroxyl of the tetrahedral carbon, and the tetrahedral
intermediate breaks down in a manner consistent with the stereoelectronic
hypothesis. The models permit the rationalization of observed subsite
preferences for substrates and may be useful in predicting subsite preferences
of other aspartic proteinases.
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