Long-range structural changes in proteinase K triggered by calcium ion removal.

The X-ray crystal structure of the subtilisin-type enzyme proteinase K at 1.5 A resolution shows that is has two binding sites for Ca2+. Scatchard analysis indicates that one Ca2+ binds tightly, with pK 7.6 x 10(-8) M-1, and the other only weakly. Although Ca2+ is not directly involved in the catalytic mechanism and is 16.6 A away from the alpha-carbon atoms of the catalytic triad Asp 39-His 69-Ser 224, the activity of proteinase K towards the synthetic substrate succinyl-Ala-Ala-Ala-p-nitroanilide drops slowly to approximately 20% of its original value when it is depleted of Ca2+. This is not due to autolysis of the enzyme. The X-ray crystal structure of Ca2+-free proteinase K shows that removal of Ca2+ from the tight binding site triggers a concerted domino-like movement of five peripheral loops and of two alpha-helices. At a distance of 25 A from this calcium-binding site, the geometry of both the secondary substrate binding site and of the catalytic triad is affected by this movement thereby reducing the activity of the enzyme.