Recombinant human cathepsin H lacking the mini chain is an endopeptidase

Human procathepsin H was expressed in the form of inclusion bodies in Escherichia coli. Following refolding and autocatalytic activation, a recombinant Cathepsin H form lacking the mini chain was produced. Removal of the mini chain completely abolished Aminopeptidase activity of the Enzyme and largely increased its endopeptidase activity (approximately 40-fold). Similarly to Cathepsin S, Bz-FVR-AMC (k(cat)/K(m) value of 1070 mM(-1) s(-1)) was found to be the preferred substrate of recombinant Cathepsin H. However, substrate inhibition was observed at a higher substrate (Z-FR-AMC, Bz-FVR-AMC) concentration. Endopeptidase activity of recombinant Cathepsin H was seen also with the protein substrate Insulin beta-chain with the major cleavage site between Glu13-Ala14. Recombinant human Cathepsin H was inhibited by chicken cystatin, stefin A, and stefin B with the K(i) values in the range of 0.05-0.1 nM, which is slightly tighter than the inhibition of purified Cathepsin H by the same inhibitors. These results thus indicate that the Cathepsin H mini chain is essential for the Aminopeptidase activity of the Enzyme but has only a minor effect on the inhibition by cystatins.