title: Activation and enzymatic characterization of recombinant human kallikrein 8.
abstract: Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidase-conjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest kcat/Km value of 20x10(3)M-1 s-1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 microM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na+ had no effect on hK8 activity, Ni2+ and Zn2+ decreased the activity and Ca2+, Mg2+, and K+ had a stimulatory effect. Ca2+ was the best activator, with an optimal concentration of approximately 10 microM.
authors.affiliation: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5G 1L5, ON, Canada.
authors.forename: Tadaaki
authors.initials: T
authors.lastname: Kishi
authors.forename: Sylvain M
authors.initials: SM
authors.lastname: Cloutier
authors.forename: Christoph
authors.initials: C
authors.lastname: Kündig
authors.forename: David
authors.initials: D
authors.lastname: Deperthes
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000187:drug effects*; D019216:Metals, Heavy / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D019216:Metals, Heavy; D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C036903:tertiary-butyloxycarbonyl-valyl-prolyl-arginyl-7-amino-4-methylcoumarin; C094799:KLK8 protein, human; D007610:Kallikreins; D002118:Calcium