Calcium binding to the class I alpha-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 (KD = 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 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 Km and kcat of 0.5 mM and 12 s-1, respectively. The Km of E526Q was greatly increased to 4 mM with a small reduction in kcat to 5 s-1 whereas the kcat values of D86N and E132Q(V) were greatly reduced (0.005-0.007 s-1) with a decrease in Km (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.
Lipari
F
F
McGill Cancer Centre, McGill University, Montréal, Québec, Canada.
Herscovics
A
A
D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D002118:Calcium