Calcium binding to the class I α-1,2-mannosidase from Saccharomyces cerevisiae occurs outside the EF hand motif

Class I alpha-1,2-mannosidases are a family of Ca2+-dependent enzymes that have been conserved through eukaryotic evolution. These enzymes contain a conserved putative EF hand Ca2+-binding motif and nine invariant acidic residues. The catalytic domain of the alpha-1,2-mannosidase from Saccharomyces cerevisiae was expressed in Pichia pastoris and was shown by atomic absorption and equilibrium dialysis to bind one Ca2+ ion with high affinity (K-D = 4 x 10(-7) M). Ca2+ protected the enzyme from thermal denaturation. Mutation of the 1st and 12th residues of the putative EF hand Ca2+ binding loop (D121N, D121A, E132Q, E132V, and D121A/E132V) had no effect on Ca2+ binding, demonstrating that the EF hand motif is not the site of Ca2+ binding. In contrast, three invariant acidic residue mutants (D275N, E279Q, and E438Q) lost the ability to bind Ca-45(2+) following nondenaturing polyacrylamide gel electrophoresis whereas D86N, E132Q, E503Q, and E526Q mutants exhibited binding of Ca-45(2+) similar to the wild-type enzyme. The wild-type enzyme had a K-m and k(cat) of 0.5 mM and 12 s(-1), respectively. The K-m of E526Q was greatly increased to 4 mM with a small reduction in k(cat) to 5 s(-1) whereas the k(cat) values of D86N and E132Q(V) were greatly reduced (0.005-0.007 s(-1)) with a decrease in K-m (0.07-0.3 mM). The E503Q mutant is completely inactive. Asp(275), Glu(279), and Glu(438) are therefore required for Ca2+ binding whereas Asp(86), Glu(132), and Glu(503) are required fur catalysis.