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...

Human platelet heparanase has been purified to homogeneity and shown to consist of two, non-covalently associated polypeptide chains of molecular masses 50 and 8 kDa. Protein sequencing provided the basis for determination of the full-length cDNA for this novel protein. Based upon this information and results from protein analysis and mass spectrometry, we propose a scheme to define the structural organization of heparanase in relation to its precursor forms, proheparanase and pre-proheparanase. The 8-and 50-kDa chains which make up the active enzyme reside, respectively, at the NH 2 -and COOH-terminal regions of the inactive precursor, proheparanase. The heparanase heterodimer is produced by excision and loss of an internal linking segment. This paper is the first to suggest that human heparanase is a two-chain enzyme.

The binding of 125I-labeled immunogenic peptides to purified Ia molecules in detergent solution was examined by equilibrium dialysis. We used the chicken ovalbumin peptide ovalbumin-(323-339)-Tyr, which is immunogenic in the BALB/c mouse and restricted to I-Ad. 125I-labeled ovalbumin-(323-339)-Tyr was shown to bind to I-Ad but not to I-Ed, I-Ek, or I-Ak. This binding was inhibited by unlabeled ovalbumin-(323-339) but not by ovalbumin-(329-339), which is the longest N-terminally truncated peptide that fails to stimulate any of the I-Ad-restricted hybridomas that have been raised to ovalbumin-(323-339)-Tyr. As a further specificity control, we also used the chicken egg lysozyme peptide Tyr-(46-61), which has recently been studied by similar methods [Babbitt, B. P., Allen, P. M., Matsueda, G., Haber, E. & Unanue, E. R. (1985) Nature (London) 317, 359-361]. We have confirmed that it bound to I-Ak but not to I-Ek, I-Ad, or I-Ed. Thus, a specific interaction between Ia and antigen that correlates with the major histocompatibility complex restriction was demonstrated, strongly arguing in favor of a determinant selection hypothesis for such restriction.

No abstract

The involvement of b-secretase (BACE1; b-site APP-cleaving enzyme) in producing the b-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APP Sw , and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2¢ sequence, …VKM-fl-DA…, was replaced by …ISY-fl-EV… (-fl-¼ b site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APP Sw peptide, SEVNL-fl-DAEFR. The expanded peptide, GLTNIKTEEISEISY-fl-EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these b-site amino acid changes were prepared as the basis for cellular assays. The APP ISYEV mutant proved to be a cellular substrate that was superior to APP Sw . The assay based on APP ISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APP ISYEV at the b-site. Insertion of the optimized ISY-fl-EV motif at either the b-site (Asp1) or b¢-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease. Keywords: Alzheimer's b-secretase, APP processing, amyloid b-peptide, BACE1, BACE2, mutant amyloid precursor protein.