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The fungal proteinase K (EC 3.4.21.14) is a very potent unusually stable member of the subtilisin family. Its X-ray structure determined at 0.15-nm resolution shows two bound Ca2+ ions. Cal isin near-ideal pentagonal bipyramidal configuration with Asp200 carboxylate and Prol75 peptide C=O in an apical, and Vai177 peptide C=O and four water molecules in an equatorial position, whereas Ca2 displays incomplete octahedral coordination with the carboxylate of Asp260, the peptide C=O of Vail6 and two water molecules. Scatchard analysis of the titration of Ca2+-free proteinase K with Ca2+ yields a single dissociation constant (7.6±2.5) × 10 −8 M associated with the tightly bound Cal whereas Ca2 is so weakly bound that it cannot be titrated. If proteinase K is depleted of Ca2+ by treatment with EDTA, followed by gel filtration, its enzymatic activity drops within 6 h to 20% of its original value, without autolysis. Addition of excess Ca2+ immediately raises the residual activity to 28%, but full activity is not achieved. Removal of Ca2+ triggers a conformational change of the substrate recognition site because there is a direct connection, via secondary structure hydrogen bonds, between the Cal binding site and the substrate-recognition site. This is indicated further by circular dichroism and fluorescence-spectroscopic data, and by reversed-phase FPLC, carried out in the presence and absence of Ca2+, but the overall structure of the enzyme is not affected. Depletion of Ca2+ also influences binding of longer peptide inhibitors of the chloromethane type, it increases the rate of autolysis after about 48 h, it reduces the thermal stability (measured by activity tests from 65 °C to 46 °C), and it enhances the deactivation by 8 M urea which inactivates to only 65%, whereas sodium dodecyl sulfate totally inactivates at a concentration of 12.5%.