Autocatalytic activation of human cathepsin K

The in vitro activation of the recombinant purified human cathepsin K (EC 3.4.22.38) was examined by mutagenesis, Cathepsin K was expressed as a secreted proenzyme using baculovirus-infected Sf21 insect cells, Spontaneous in vitro activation of procathepsin K occurred at pH 4 and was catalyzed by exogenous mature cathepsin K, Three intermediates were identified as resulting from cleavages after Glu(19), Ser(98), and Glu(110), The mature enzyme was composed of mixture of enzymes with N termini of Gly(113), Arg(114), and Ala(115) with varying ratios depending on the preparation, Molecular weight determinations were consistent with the absence of carbohydrate in the mature protein, while electrospray mass spectroscopy indicated that six of the eight cysteine residues were in disulfide linkage, and that the protein had Met(329) as the C-terminal residue. A mutant was constructed in which the active site Cys(139) was changed to Ser. [Ser(139),Ala(163)]Procathepsin K (containing mutation C139S,S163A) failed to spontaneously process and was only partially processed in the presence of 1% exogenous wild-type mature cathepsin K forming intermediates, which were identical to those observed in the activation of wild-type, [Ser(139),Ala(163)]Procathepsin K could be fully processed to mature enzyme by including one equivalent of wild-type procathepsin K in the activation mixture, These results indicated that in vitro activation of the procathepsin K was an autocatalytic process.