The role of metal Ions in the structure and function of Glucose dehydrogenase in Escherichia Coli

Abstract

Glucose dehydrogenase (GDH) is a quinoprotein, containing the prosthetic group pyrroloquinoline quinone (PQQ), that catalyses the oxidation of D-glucose to D-gluconate in the periplasm, transferring electrons to ubiquinone in the membrane. It is a single polypeptide (87kDa) that is attached to the inner membrane of E. coli. It occurs as an apoenzyme because E. coli cannot synthesise PQQ but holoGDH can be formed by incubation with Mg²⁺ and PQQ. Analysis of the amino acid sequence predicts that it has a transmembrane domain and a periplasmic superbarrel domain.

The principle aim of this thesis was to study the role of divalent metal ions in the structure and function of GDH. All other quinoproteins contain Ca²⁺ at the active site where it plays a structural and catalytic role. However, it is unclear whether Mg²⁺ or Ca²⁺ forms part of the active site of GDH because Mg²⁺ (and not Ca²⁺) is required for reconstitution of active enzyme from PQQ and apoGDH. To investigate this, amino acids that have been predicted to bind to the active site metal ion were mutated (D354N, N355D and T424N).

Characterisation of WT-GDH, D354N-GDH, N355D-GDH, D354N/N355D-GDH and T424N-GDH suggested that the metal ion added in the reconstitution process becomes incorporated into the active site and that in WT-GDH, Mg²⁺ replaces the Ca²⁺ seen in all other quinoproteins. WT-GDH could be reconstituted with Mg²⁺ but Ca²⁺, Sr²⁺ and Ba²⁺ inhibited this process by competing for the same binding site. Mutant GDHs had an altered specificity; usually they could only be reconstituted with Ca²⁺, Sr²⁺ or Ba²⁺. ApoGDH contained 3.5 molecules of Ca²⁺ and 0.82 molecules of Mg²⁺ per molecule of enzyme. This suggested that active site Ca²⁺ was already bound, but this is perhaps unlikely as eventually half the bonds to this active site metal ion are provided by PQQ, which is not present until the reconstitution process.

Mutant GDHs had a lower affinity for substrates as well in addition to the altered specificity for metal ions, suggesting that Ca²⁺, Sr²⁺ and Ba²⁺, but not Mg²⁺ are able to stabilise a conformation of the active site suitable for substrate binding. Affinity for substrates was lower with Sr²⁺ and Ba⁺-GDH than with Ca²⁺-GDH suggesting larger metal ions are better for stabilisation than smaller metal ions.

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