Calcium Binding to the Class I α-1,2-Mannosidase fromSaccharomycescerevisiaeOccurs Outside the EF Hand Motif

Abstract

Class I α-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 α-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 × 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 ⁴⁵Ca²⁺ following nondenaturing polyacrylamide gel electrophoresis whereas D86N, E132Q, E503Q, and E526Q mutants exhibited binding of ⁴⁵Ca²⁺ 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. Asp²⁷⁵, Glu²⁷⁹, and Glu⁴³⁸ are therefore required for Ca²⁺ binding whereas Asp⁸⁶, Glu¹³², and Glu⁵⁰³ are required for catalysis.