Relevance of Arg457 for the nucleotide affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Iván Tobar, Fernando D. González-Nilo, Ana M. Jabalquinto, Emilio Cardemil

Resultado de la investigación: Article

1 Cita (Scopus)

Resumen

Phosphoenolpyruvate carboxykinases catalyze one of the first steps in the biosynthesis of glucose and depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. The Saccharomyces cerevisiae enzyme has a marked preference for ADP (or ATP) over other nucleotides. Homology models of the enzyme in complex with ADP or ATP show that the guanidinium group of Arg457 is close to the adenine base, suggesting that this group might be involved in the stabilization of the nucleotide substrate. To evaluate this we have performed the mutation Arg457Met, replacing the positively charged guanidinium group by a neutral residue. The mutated enzyme retained the structural characteristics of the wild-type protein. Fluorescence titration experiments showed that mutation causes a loss of 1.7 kcal mol-1 in the binding affinity of the enzyme for ADPMn. Similarly, kinetic analyses of the mutated enzyme showed 50-fold increase in Km for ADPMn, with minor alterations in the other kinetic parameters. These results show that Arg457 is an important factor for nucleotide binding by S. cerevisiae PEP carboxykinase.

Idioma originalEnglish
Páginas (desde-hasta)1883-1889
Número de páginas7
PublicaciónInternational Journal of Biochemistry and Cell Biology
Volumen40
N.º9
DOI
EstadoPublished - 29 feb 2008

Huella dactilar

Phosphoenolpyruvate
Yeast
Saccharomyces cerevisiae
Nucleotides
Enzymes
Guanidine
Adenosine Diphosphate
Adenosine Triphosphate
Mutation
Guanine Nucleotides
Adenine Nucleotides
Biosynthesis
Adenine
Substrates
Titration
Kinetic parameters
Stabilization
Fluorescence
Glucose
Kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Citar esto

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title = "Relevance of Arg457 for the nucleotide affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase",
abstract = "Phosphoenolpyruvate carboxykinases catalyze one of the first steps in the biosynthesis of glucose and depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. The Saccharomyces cerevisiae enzyme has a marked preference for ADP (or ATP) over other nucleotides. Homology models of the enzyme in complex with ADP or ATP show that the guanidinium group of Arg457 is close to the adenine base, suggesting that this group might be involved in the stabilization of the nucleotide substrate. To evaluate this we have performed the mutation Arg457Met, replacing the positively charged guanidinium group by a neutral residue. The mutated enzyme retained the structural characteristics of the wild-type protein. Fluorescence titration experiments showed that mutation causes a loss of 1.7 kcal mol-1 in the binding affinity of the enzyme for ADPMn. Similarly, kinetic analyses of the mutated enzyme showed 50-fold increase in Km for ADPMn, with minor alterations in the other kinetic parameters. These results show that Arg457 is an important factor for nucleotide binding by S. cerevisiae PEP carboxykinase.",
keywords = "π-π stacking interactions, Cation-π interactions, Nucleotide affinity, Phosphoenolpyruvate carboxykinase, Saccharomyces cerevisiae",
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Relevance of Arg457 for the nucleotide affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase. / Tobar, Iván; González-Nilo, Fernando D.; Jabalquinto, Ana M.; Cardemil, Emilio.

En: International Journal of Biochemistry and Cell Biology, Vol. 40, N.º 9, 29.02.2008, p. 1883-1889.

Resultado de la investigación: Article

TY - JOUR

T1 - Relevance of Arg457 for the nucleotide affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

AU - Tobar, Iván

AU - González-Nilo, Fernando D.

AU - Jabalquinto, Ana M.

AU - Cardemil, Emilio

PY - 2008/2/29

Y1 - 2008/2/29

N2 - Phosphoenolpyruvate carboxykinases catalyze one of the first steps in the biosynthesis of glucose and depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. The Saccharomyces cerevisiae enzyme has a marked preference for ADP (or ATP) over other nucleotides. Homology models of the enzyme in complex with ADP or ATP show that the guanidinium group of Arg457 is close to the adenine base, suggesting that this group might be involved in the stabilization of the nucleotide substrate. To evaluate this we have performed the mutation Arg457Met, replacing the positively charged guanidinium group by a neutral residue. The mutated enzyme retained the structural characteristics of the wild-type protein. Fluorescence titration experiments showed that mutation causes a loss of 1.7 kcal mol-1 in the binding affinity of the enzyme for ADPMn. Similarly, kinetic analyses of the mutated enzyme showed 50-fold increase in Km for ADPMn, with minor alterations in the other kinetic parameters. These results show that Arg457 is an important factor for nucleotide binding by S. cerevisiae PEP carboxykinase.

AB - Phosphoenolpyruvate carboxykinases catalyze one of the first steps in the biosynthesis of glucose and depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. The Saccharomyces cerevisiae enzyme has a marked preference for ADP (or ATP) over other nucleotides. Homology models of the enzyme in complex with ADP or ATP show that the guanidinium group of Arg457 is close to the adenine base, suggesting that this group might be involved in the stabilization of the nucleotide substrate. To evaluate this we have performed the mutation Arg457Met, replacing the positively charged guanidinium group by a neutral residue. The mutated enzyme retained the structural characteristics of the wild-type protein. Fluorescence titration experiments showed that mutation causes a loss of 1.7 kcal mol-1 in the binding affinity of the enzyme for ADPMn. Similarly, kinetic analyses of the mutated enzyme showed 50-fold increase in Km for ADPMn, with minor alterations in the other kinetic parameters. These results show that Arg457 is an important factor for nucleotide binding by S. cerevisiae PEP carboxykinase.

KW - π-π stacking interactions

KW - Cation-π interactions

KW - Nucleotide affinity

KW - Phosphoenolpyruvate carboxykinase

KW - Saccharomyces cerevisiae

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