A peptide inhibitor, having the sequence D-His-Pro-Phe-His-PheI[CH2-NH]Phe-Val-Tyr, with a reduced bond between the two adjacent phenylalanines, has been diffused into crystals of the aspartic proteinase from Rhizopus chinensis (rhizopuspepsin, EC 3.4.23.6). X-ray diffraction data to 1.8-A resolution have been collected on the complex, which has been subjected to restrained least-squares refinement to an R-factor (R = Z;|1FO1 -FII[IFOI, where IFOI and JFJ are the observed and calculated structure factor amplitudes, respectively) of 14.7%. The inhibitor lies within the major groove of the enzyme and is clearly defined with the exception of the amino-terminal D-histidine and the carboxyl-terminal tyrosine.The reduced peptide bond is located in the active site with close contacts to the two catalytic aspartyl groups. The active-site water molecule that is held between the two carboxyl groups is displaced by the inhibitor, as are a number of other water molecules seen in the binding groove of the native enzyme. A mechanism of action for this class of enzymes is proposed from these results.The aspartic proteinases, which include the mammalian enzymes pepsin, gastricsin, chymosin, cathepsin D, and renin, as well as a number of microbial and plant enzymes, form a class of digestive enzymes having several common properties. They are optimally active at acidic pH, have two aspartic acids at the active site, and are all inactivated by certain inhibitors, such as pepstatin. In addition, they all display subsite specificities extending for several residues on either side of the scissile bond. Detailed x-ray analyses for four of these enzymes-namely, pepsin and three fungal enzymes, penicillopepsin, endothiapepsin and rhizopuspepsin [refs. 1, 2 (pp. 137-150), 3-8] have shown very similar three-dimensional structures; this similarity is particularly evident in the enzyme active sites. The mechanism of action of these proteinases has been extensively discussed (for review, see ref.2). Earlier studies provided no compelling evidence for the formation of a covalent intermediate during catalysis (9,10). Consequently, recent mechanistic hypotheses based on the three-dimensional structures of these enzymes have invoked nucleophilic attack by a water molecule on the carbonyl carbon, with possible intervention of the carboxyl groups of the enzyme in transferring a proton to the substrate amino group [refs. 2 (pp. 189-195), 11, 12]. However, the identity of this water molecule remains unresolved.We describe an 1.8-A analysis of the complex of rhizopuspepsin with a reduced peptide inhibitor of the sequence D-His-Pro-Phe-His-PheT[CH2-NH]Phe-Val-Tyr [nomenclature of Spatola (13)] and relate these results to a mechanism of action for aspartic proteinases.
MATERIALS AND METHODSCrystals of the native enzyme were grown as described (8). Intensity data were collected using the Mark II multiwire area detector system at the University of California at San Diego [Resource for Protein Crystallography, National Institutes of Health Grant...
