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Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase: The Relevance of Glu299 and Leu460 for Nucleotide Binding

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Abstract

A homology model of Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase (ATP + oxaloacetate ⇄ ADP + PEP + CO2) in complex with its substrates shows that the isobutyl group of Leu460 is in close proximity to the adenine ring of the nucleotide, while the carboxyl group of Glu299 is within hydrogen-bonding distance of the ribose 2′OH. The Leu460Ala mutation caused three-fold and seven-fold increases in the K m for ADPMn− and ATPMn2−, respectively, while the Glu299Ala mutation had no effect. Binding studies showed losses of approximately 2 kcal mol−1 in the nucleotide binding affinity due to the Leu460Ala mutation and no effect for the Glu299Ala mutation. PEP carboxykinase utilized 2′deoxyADP and 2′deoxyATP as substrates with kinetic and equilibrium dissociation constants very similar to those of ADP and ATP, respectively. These results show that the hydrophobic interaction between Leu460 and the adenine ring of the nucleotide significantly contributed to the nucleotide affinity of the enzyme. The 2′deoxy nucleotide studies and the lack of an effect of the Glu299Ala mutation in nucleotide binding suggest that the possible hydrogen bond contributed by Glu299 and the ribose 2′OH group may not be relevant for nucleotide binding.

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Abbreviations

CD:

Circular dichroism.

HEPES:

N-(2-hydroxyethyl) piperazine-N′-2(ethanesulfonic acid).

MOPS:

3-(N-morpholino) propanesulfonic acid.

OAA:

Oxaloacetate.

P-pyridoxyl:

Phosphopyridoxyl.

PEP:

Phosphoenolpyruvate.

PLP:

Pyridoxal 5′-phosphate.

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Acknowledgments

Estela Pérez holds a postdoctoral fellowship from the Universidad de Santiago de Chile. We thank Mr. Rodrigo Espinoza for help during this work. This research was supported by research grant FONDECYT 1070202.

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Authors and Affiliations

  1. Facultad de Química y Biología, Universidad de Santiago de Chile, Av. B. O’Higgins 3363, Santiago, 9170022, Chile

    Estela Pérez & Emilio Cardemil

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  1. Estela Pérez
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  2. Emilio Cardemil
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Correspondence to Emilio Cardemil.

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Pérez, E., Cardemil, E. Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase: The Relevance of Glu299 and Leu460 for Nucleotide Binding. Protein J 29, 299–305 (2010). https://doi.org/10.1007/s10930-010-9252-6

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