Prime region subsite specificity characterization of human cathepsin D: the dominant role of position 128.

In order to contribute to our understanding of cathepsin D (CatD) active site specificity, two series of chromogenic octapeptides with systematic substitutions in positions P2' and P3' were synthesized. This panel was characterized with native human liver cathepsin D (nHuCatD) and yielded information concerning specificity trends within the S2' and S3' subsites. The pepstatin inhibited crystal structure of nHuCatD (Baldwin et al., 1993) was then utilized in conjunction with these subsite preference data to identify residues suspected of contributing to "prime" side subsite specificity. These residues were targeted for site-directed mutagenesis using the re-engineered recombinant model, "short" pseudocathepsin D (Beyer & Dunn, 1996). As a result of these analyses it was determined that prime region subsites do contribute to the unique specificity of human CatD. Furthermore, it was ascertained that the poly-proline loop does not have an active role in S3' subsite specificity. Lastly, it appears that Ile128 has a dominant role on S2' subsite specificity whereas Val130 does not.

Self-activation of recombinant human lysosomal procathepsin D at a newly engineered cleavage junction, "short" pseudocathepsin D.

To obtain a recombinant model of human cathepsin D with kinetic properties that are identical with native human liver enzyme, we have addressed the significant differences in structure and catalytic function between naturally occurring enzyme and bacterially derived pseudocathepsin D. Human procathepsin D was expressed in a baculovirus system to obtain correctly folded, glycosylated enzyme that upon acidification completely converts to the active intermediate, pseudocathepsin D. The oligosaccharide moieties of this recombinant enzyme contributed to about 5% of the apparent molecular mass of the enzyme, and the carbohydrate composition was quite similar to the native material. However, specificity constants (kcat/Km) of this glycosylated pseudoform for several synthetic chromogenic substrates were considerably less (33%-50%) than those for the native enzyme and were virtually identical with those observed with nonglycosylated pseudocathepsin D. A cleavable junction suitable for self-processing at the normal maturation point of human cathepsin D was engineered into procathepsin D according to known specificity requirements of this enzyme, and the construct was expressed using baculovirus. Following experiments that demonstrated that the new proenzyme failed to process to the expected point, the new cleavage junction was moved 6 residues toward the amino terminus of procathepsin D and expressed in Escherichia coli. After refolding, the protein containing the newly engineered junction self-processed, generating a shortened mutant form of pseudocathepsin D that is 6 residues longer at the amino terminus than the native material. The kinetic properties of this newly engineered pseudoform proved to be identical with those of the native enzyme, thus establishing an improved recombinant model for this important aspartic proteinase.

Effect of naturally occurring active site mutations on hepatitis C virus NS3 protease specificity.

A comparison of the DNA sequences from all available genotypes of HCV indicate that the active site residues of the NS3 protease are strictly conserved with the exception of positions 123 and 168, which border the S(4) subsite. In genotype 3, the canonic arginine and aspartic acid have been replaced with threonine and glutamine, respectively. To determine if these differences contribute to an altered specificity, we characterized single-chain NS3 proteases from strains 1a, 1b, and 3a with peptide substrates and product inhibitors on the basis of the natural cleavage junction sequences, in addition to polyprotein substrates derived from the 1a strain. No statistically significant differences in specificity were observed. To demonstrate that the active sites were actually different, we generated and evaluated peptide substrates with unnatural extended side-chains. These studies confirmed that there are measurable differences between the NS3 proteases of genotypes 1 and 3. Specifically, a 5-fold difference in K(i) was observed between the proteases from genotypes 1 and 3 when a D-Glu occupied P(5), and a 30-fold difference was seen when this position contained a D-homoglutamate. The contribution of residues 123 and 168 toward the altered specificity was then evaluated individually by site-directed mutagenesis. These mutants showed that potency differences within this series could be attributed to the residue that occupied position 123 of the protease. Modeling these unnatural substrate/mutant protease interactions, on the basis of cocrystal structures of enzyme-substrate complexes, provides a structural basis for these observations. Proteins 2001;43:82-88.

A continuous spectrophotometric assay for the hepatitis C virus serine protease.

The hepatitis C virus (HCV) encodes a chymotrypsin-like serine protease responsible for the processing of HCV nonstructural proteins and which is a promising target for antiviral intervention. Its relatively low catalytic efficiency has made standard approaches to continuous assay development only modestly successful. In this report, four continuous spectrophotometric substrates suitable for both high-throughput screening and detailed kinetic analysis are described. One of these substrates, Ac-DTEDVVP(Nva)-O-4-phenylazophenyl ester, is hydrolyzed by HCV protease with a second-order rate constant (kcat/Km) of 80,000 +/- 10,000 M-1 s-1. Together with its negligible rate of nonenzymatic hydrolysis under assay conditions (0.01 h-1), analysis of as little as 2 nM protease can be completed in under 10 min.