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

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Abstract
The X-ray crystal structure of the subtilisin-type enzyme proteinase K at 1.5 Å resolution1 shows that is has two binding sites for Ca2+. Scatchard analysis2 indicates that one Ca2+ binds tightly, with pK 7.6 × 10−8 M−1, and the other only weakly. Although Ca2+ is not directly involved in the catalytic mechanism and is 16.6 Å away from the α-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 ∼ 20% of its original value when it is depleted of Ca2+. This is not due to autolysis of the enzyme2. 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 α-helices. At a distance of 25 Å 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.

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