CRYSTAL-STRUCTURE OF PORCINE CATHEPSIN-H DETERMINED AT 2.1 ANGSTROM RESOLUTION - LOCATION OF THE MINI-CHAIN C-TERMINAL CARBOXYL GROUP DEFINES CATHEPSIN-H AMINOPEPTIDASE FUNCTION

Background: Cathepsin H is a lysosomal cysteine protease, involved in intracellular protein degradation. It is the only known mono-aminopept idase in the papain-like family and is reported to be involved in tumo r metastasis. The cathepsin H structure was determined in order to inv estigate the structural basis for its aminopeptidase activity and thus to provide the basis for structure-based design of synthetic inhibito rs. Results: The crystal structure of native porcine cathepsin H was d etermined at 2.1 Angstrom resolution, The structure has the typical pa pain-family fold. The so-called mini-chain, the octapeptide EPQNCSAT, is attached via a disulfide bond to the body of the enzyme and bound i n a narrowed active-site cleft, in the substrate-binding direction. Th e mini-chain fills the region that in related enzymes comprises the no n-primed substrate-binding sites from S2 backwards. Conclusions: The c rystal structure of cathepsin H reveals that the mini-chain has a defi nitive role in substrate recognition and that carbohydrate residues at tached to the body of the enzyme are involved in positioning the mini- chain in the active-site cleft. Modeling of a substrate into the activ e-site cleft suggests that the negatively charged carboxyl group of th e C terminus of the mini-chain acts as an anchor for the positively ch arged N-terminal amino group of a substrate. The observed displacement s of the residues within the active-site cleft from their equivalent p ositions in the papain-like endopeptidases suggest that they form the structural basis for the positioning of both the mini-chain and the su bstrate, resulting in exopeptidase activity.