CATION-BINDING TO A BACILLUS (1,3-1,4)-BETA-GLUCANASE - GEOMETRY, AFFINITY AND EFFECT ON PROTEIN STABILITY

The hybrid Bacillus (1,3-1,4)-beta-glucanase H(A16-M), consisting of 1 6 N-terminal amino acids derived from the mature form of the B. amylol iquefaciens enzyme and of 198 C-proximal amino acids from the B. macer ans enzyme, binds a calcium ion at a site at its molecular surface rem ote from the active center [T. Keitel, O. Simon, R. Borriss and U. Hei nemann (1993) Proc. Natl Acad. Sci. USA 90, 5287-5291]. X-ray diffract ion 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 coo rdination sphere, two of which are from water molecules, sodium is fiv efold 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 bind ing stabilizes the native three-dimensional structure of the protein a s shown by guanidinium chloride unfolding and thermal inactivation exp eriments. The enhanced enzymic activity of Bacillus beta-glucanases at elevated temperatures in the presence of calcium ions is attributed t o a general stabilizing effect by the cation.