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

Tobar I.; Gonzalez-Nilo, FD; Jabalquinto, AM; Cardemil, E

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. © 2008 Elsevier Ltd. All rights reserved.

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Título según WOS: Relevance of Arg457 for the nucleotide affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase
Título según SCOPUS: Relevance of Arg457 for the nucleotide affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase
Título de la Revista: INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
Volumen: 40
Número: 9
Editorial: PERGAMON-ELSEVIER SCIENCE LTD
Fecha de publicación: 2008
Página de inicio: 1883
Página final: 1889
Idioma: English
URL: http://linkinghub.elsevier.com/retrieve/pii/S1357272508000423
DOI:

10.1016/j.biocel.2008.01.032

Notas: ISI, SCOPUS