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Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.
Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav.
Afiliación
  • Isaksen GV; Centre for Theoretical and Computational Chemistry, Department of Chemistry, Faculty of Science and Technology, University of Tromsø , N9037 Tromsø, Norway.
  • Hopmann KH; Centre for Theoretical and Computational Chemistry, Department of Chemistry, Faculty of Science and Technology, University of Tromsø , N9037 Tromsø, Norway.
  • Åqvist J; Department of Cell & Molecular Biology, Uppsala University , SE-75124 Uppsala, Sweden.
  • Brandsdal BO; Centre for Theoretical and Computational Chemistry, Department of Chemistry, Faculty of Science and Technology, University of Tromsø , N9037 Tromsø, Norway.
Biochemistry ; 55(14): 2153-62, 2016 Apr 12.
Article en En | MEDLINE | ID: mdl-26985580
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Modelos Moleculares / Adenosina / Purina-Nucleósido Fosforilasa / Proteínas Mutantes / Guanosina / Inosina Límite: Humans Idioma: En Revista: Biochemistry Año: 2016 Tipo del documento: Article País de afiliación: Noruega Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Modelos Moleculares / Adenosina / Purina-Nucleósido Fosforilasa / Proteínas Mutantes / Guanosina / Inosina Límite: Humans Idioma: En Revista: Biochemistry Año: 2016 Tipo del documento: Article País de afiliación: Noruega Pais de publicación: Estados Unidos