Activity:
Activity of mature protein was measured in 50mM MES, pH 5.5, containing 2.5mM EDTA and 5mM DTT. Assay was initiated by addition of substrate Z-Phe-Arg-AMC (Prod. No. ALX-260-131) to a final concentration of 100µM (stock solution 10mM in DMSO). Activity was monitored by the increase of fluorescence accompanying release of the product 7-amino-4-methylcoumarin (AMC) (excitation wavelength of 360nm; emission wavelength of 460nm) (see [2]).

Activation:
Activate by adjusting procathepsin K to pH 4.0 by adding an equal volume of 100mM sodium acetate, pH 3.9, containing 10mM DTT, 5mM EDTA and incubate for 40 min. at room temperature. MMTS (methyl methanethiosulfonate) modifies cysteines by attaching its relatively small, uncharged thiomethyl blocking group to reactive sulfhydryl groups (see [1]). This reversible reaction arrests the autoproteolytical process. Activity can be restored to nearly unmodified level by adding L-cysteine (~3M excess over MMTS).

Inhibitors:
Cathepsin K is inhibited by leupeptin (Prod. No. ALX-260-009) (IC₅₀=70nM), E-64 (IC₅₀=5nM) or cystatine. Minimal effects exhibit pepstatin, phenylmethylsulfonyl fluoride (Prod. No. ALX-270-184), inhibitors of aspartyl and serine proteases. No inhibition was observed by addition of EDTA or phenanthroline, classical inhibitors of metalloproteases.

Swiss-Prot link P43235: Cathepsin K (human) (precursor)