Cation binding to a Bacillus (1,3–1,4)‐β‐glucanase Geometry, affinity and effect on protein stability

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Abstract
The hybrid Bacillus (1,3–1,4)‐β‐glucanase H(A16‐M), consisting of 16 N‐terminal amino acids derived from the mature form of the B. amyloliquefaciens enzyme and of 198 C‐proximal amino acids from the B. macerans enzyme, binds a calcium ion at a site at its molecular surface remote from the active center [T. Keitel, O. Simon, R. Borriss & U. Heinemann (1993) Proc. Natl Acad. Sci. USA 90, 5287–5291]. X‐ray diffraction analysis at 0.22‐nm resolution of crystals grown in the absence of calcium and in the presence of EDTA shows this site to be occupied by a sodium ion. Whereas the calcium ion has six oxygen atoms in its coordination sphere, two of which are from water molecules, sodium is fivefold coordinated with a fifth ligand belonging to a symmetry‐related protein molecule in the crystal lattice. The affinity of H(A16‐M) for calcium over sodium has been determined calorimetrically. Calcium binding stabilizes the native three‐dimensional structure of the protein as shown by guanidinium chloride unfolding and thermal inactivation experiments. The enhanced enzymic activity of Bacillusβ‐glucanases at elevated temperatures in the presence of calcium ions is attributed to a general stabilizing effect by the cation.