title: The metal ion in the active site of the membrane glucose dehydrogenase of Escherichia coli.
abstract: All pyrroloquinoline quinone (PQQ)-containing dehydrogenases whose structures are known contain Ca(2+) bonded to the PQQ at the active site. However, membrane glucose dehydrogenase (GDH) requires reconstitution with PQQ and Mg(2+) ions (but not Ca(2+)) for activity. To address the question of whether the Mg(2+) replaces the usual active site Ca(2+) in this enzyme, mutant GDHs were produced in which residues proposed to be involved in binding metal ion were modified (D354N-GDH and N355D-GDH and D354N-GDH/N355D-GDH). The most remarkable observation was that reconstitution with PQQ of the mutant enzymes was not supported by Mg(2+) ions as in the wild-type GDH, but it could be supported by Ca(2+), Sr(2+) or Ba(2+) ions. This was competitively inhibited by Mg(2+). This result, together with studies on the kinetics of the modified enzymes have led to the conclusion that, although a Ca(2+) ion is able to form part of the active site of the genetically modified GDH, as in all other PQQ-containing quinoproteins, a Mg(2+) ion surprisingly replaces Ca(2+) in the active site of the wild-type GDH.
authors.affiliation: Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton S016 7PX, Hants, UK.
authors.forename: Peter L
authors.initials: PL
authors.lastname: James
authors.forename: Christopher
authors.initials: C
authors.lastname: Anthony
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002462:Cell Membrane / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005948:Glucose Dehydrogenases / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D005948:Glucose Dehydrogenases; C033955:glucose dehydrogenase (pyrroloquinoline-quinone); D008274:Magnesium; D002118:Calcium