The metal ion in the active site of the membrane glucose dehydrogenase of Escherichia coli

All pyrroloquinoline quinone (PQQ)-containing dehydrogenases whose structures are known contain Ca2+ bonded to the PQQ at the active site. However, membrane glucose dehydrogenase (GDH) requires reconstitution with PQQ and Mg2+ ions (but not Ca2+) for activity. To address the question of whether the Mg2+ replaces the usual active site Ca2+ 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 Mg2+ ions as in the wild-type GDH, but it could be supported by Ca2+, Sr2+ or Ba2+ ions. This was competitively inhibited by Mg2+. This result, together with studies on the kinetics of the modified enzymes have led to the conclusion that, although a Ca2+ ion is able to form part of the active site of the genetically modified GDH, as in all other PQQ-containing quinoproteins, a Mg2+ ion surprisingly replaces Ca2+ in the active site of the wildtype GDH. (C) 2003 Elsevier Science B.V. All rights reserved.