Nucleotide specificity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: Kinetics, fluorescence spectroscopy, and molecular simulation studies

José M. Villarreal, Claudia Bueno, Felipe Arenas, Ana M. Jabalquinto, Fernando D. González-Nilo, María V. Encinas, Emilio Cardemil

Resultado de la investigación: Contribución a una revistaArtículo

6 Citas (Scopus)

Resumen

Phosphoenolpyruvate carboxykinases, depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. In this work, analyses of the substrate specificity of the Saccharomyces cerevisiae ATP-dependent enzyme have been carried out. Kinetics studies gave relative values of kcat/Km for the nucleoside triphosphate complexes in the order ATP ≫ GTP > ITP > UTP > CTP. For the nucleoside diphosphate complexes the order is ADP ≫; GDP > IDP ≅ UDP > CDP. This shows that the enzyme has a strong preference for ADP (or ATP) over other nucleotides, being this preference about an order of magnitude higher for the diphosphorylated than for the triphosphorylated nucleosides. The calculated binding free energies (kcal mol-1) at 25°C are 7.39 and 6.51 for ATP and ADP, respectively. These values decrease with the nucleotide structure in the same order than the kinetic specificity. The binding energy for any triphosphorylated nucleoside is more favourable than for the corresponding diphosphorylated compound, showing the relevance of the P γ for nucleotide binding. Homology models of the adenine and guanine nucleotides in complex with the enzyme show that the base adopts a similar conformation in the diphosphorylated nucleosides while in the triphosphorylated nucleosides the sugar-base torsion angle is 61° for ATP and -53° for GTP. Differences are also noted in the distance between P β and Mn2+ at site 1. This distance is almost the same in the ATP, GTP, and UTP complexes, however in the ADP, GDP and UDP complexes it is 2.9, 5.1, and 7 Å, respectively. Experimental data obtained with a Thr463Ala mutant enzyme agree with molecular simulation predictions. The results here presented are discussed in terms of the proposed interactions of the nucleotides with the protein.

Idioma originalInglés
Páginas (desde-hasta)576-588
Número de páginas13
PublicaciónInternational Journal of Biochemistry and Cell Biology
Volumen38
N.º4
DOI
EstadoPublicada - 26 ene 2006

Áreas temáticas de ASJC Scopus

  • Bioquímica
  • Biología celular

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