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Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.
Pedroso, Marcelo M; Ely, Fernanda; Carpenter, Margaret C; Mitic, Natasa; Gahan, Lawrence R; Ollis, David L; Wilcox, Dean E; Schenk, Gerhard.
Afiliación
  • Pedroso MM; School of Chemistry and Molecular BioSciences, The University of Queensland , St Lucia, QLD 4072, Australia.
  • Ely F; School of Chemistry and Molecular BioSciences, The University of Queensland , St Lucia, QLD 4072, Australia.
  • Carpenter MC; Department of Chemistry, Dartmouth College , Hanover, New Hampshire 03755, United States.
  • Mitic N; Department of Chemistry, National University of Ireland-Maynooth , Maynooth, County Kildare, Ireland.
  • Gahan LR; School of Chemistry and Molecular BioSciences, The University of Queensland , St Lucia, QLD 4072, Australia.
  • Ollis DL; Research School of Chemistry, Australian National University , Canberra, ACT 0200, Australia.
  • Wilcox DE; Department of Chemistry, Dartmouth College , Hanover, New Hampshire 03755, United States.
  • Schenk G; School of Chemistry and Molecular BioSciences, The University of Queensland , St Lucia, QLD 4072, Australia.
Biochemistry ; 56(26): 3328-3336, 2017 07 05.
Article en En | MEDLINE | ID: mdl-28562023
Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its ß site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the ß site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the ß site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the ß site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Bacterianas / Cobalto / Hidrolasas Diéster Fosfóricas / Enterobacter aerogenes / Manganeso Idioma: En Revista: Biochemistry Año: 2017 Tipo del documento: Article País de afiliación: Australia Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Bacterianas / Cobalto / Hidrolasas Diéster Fosfóricas / Enterobacter aerogenes / Manganeso Idioma: En Revista: Biochemistry Año: 2017 Tipo del documento: Article País de afiliación: Australia Pais de publicación: Estados Unidos