<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Phosphoenolpyruvate Carboxykinase: The Relevance of Glu299 and Leu460 for Nucleotide Binding |
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Authors: | Estela Pérez Emilio Cardemil |
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Institution: | 1.Facultad de Química y Biología,Universidad de Santiago de Chile,Santiago,Chile |
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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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