Crystal structure of porcine cathepsin H determined at 2.1 å resolution: location of the mini-chain C-terminal carboxyl group defines cathepsin H aminopeptidase function

Abstract

The crystal structure of cathepsin H reveals that the mini-chain has a definitive role in substrate recognition and that carbohydrate residues attached to the body of the enzyme are involved in positioning the mini-chain in the active-site cleft. Modeling of a substrate into the active-site cleft suggests that the negatively charged carboxyl group of the C terminus of the mini-chain acts as an anchor for the positively charged N-terminal amino group of a substrate. The observed displacements of the residues within the active-site cleft from their equivalent positions in the papain-like endopeptidases suggest that they form the structural basis for the positioning of both the mini-chain and the substrate, resulting in exopeptidase activity.

Alterations in cathepsin H activity and protein patterns in human colorectal carcinomas

“…Cathepsin H enzyme activity was also measured in the presence and absence of 100 µM E-64 with no inhibition observed in either normal or cancer tissues, again demonstrating the specificity of our assay for cathepsin H activity. E-64 is a strong inhibitor of the majority of cysteine proteinases, with the exception of the cysteine proteinase cathepsin H, as reported in biochemical studies (Katunuma and Kominami, 1995) and more recently confirmed by the publication of the crystal structure of porcine cathepsin H (Guncar et al, 1998).…”

“…The activity of cathepsin H was determined against the specific synthetic substrate L-Arg-MNA (Sigma; and Enzyme System Products, Dublin, CA, USA) to take advantage of the specific aminopeptidase activity of cathepsin H, that distinguishes it from other cysteine lysosomal enzymes, such as cathepsins B and L (Schwartz and Barrett, 1980;Guncar et al, 1998). Tissue extracts (10-20 µl per assay) were pre-incubated in assay buffer (0.1 M MES-EDTA buffer, 1 mM dithiothreitol (DTT), pH 6.8) at 37°C for 5 min, using a modification of methods described by Schwartz and Barrett (1980).…”

“…These proteinases have been viewed traditionally as simple mediators of lysosomal protein degradation (Chapman et al, 1997), although studies have also demonstrated their temporal regulation during development (Mathur et al, 1997) and their specificity of expression with cell type and function (Taniguchi et al, 1993). Cathepsin H is easily distinguished from cathepsins B and L by its unique aminopeptidase activity (Guncar et al, 1998) while the latter are better described as endopeptidases .…”

Free kick instead of cross-validation in maximum-likelihood refinement of macromolecular crystal structures

“…To address the convergence and robustness of refinement, we chose five starting cases of molecular-replacement solutions of the crystal structures of cathepsin H (PDB entry 8pch; Gunčar et al, 1998) Kim et al, 2006). The structures of actinidin (Baker, 1980), Crotalus atrox phospholipase A 2 (Keith et al, 1981), stefin B (Stubbs et al, 1990), thaumatin (Ko et al, 1994) and choline acetyltransferase (Cai et al, 2004) were used as the search models (Pražnikar et al, 2009).…”

Mouse stefins A1 and A2 (Stfa1 and Stfa2) differentiate between papain‐like endo‐ and exopeptidases

“…These features compete with inhibitors for binding into the same sites on the surface of target proteases. According to the model of cystatin-papain-like protease complex N-terminal trunk competes with the features of aminopeptidases: the mini-chain of cathepsin H [20] and the exclusion domain residues of cathepsin C [21], whereas the second hairpin loop competes for binding with the occluding loop of carboxypeptidase cathepsin B [22].…”