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

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.