Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: the relevance of Glu299 and Leu460 for nucleotide binding.
A homology model of Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase (ATP + oxaloacetate right arrow over left arrow ADP + PEP + CO(2)) 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 ATPMn(2-), 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.
Facultad de Química y Biología, Universidad de Santiago de Chile, Av. B. O'Higgins 3363, Santiago, Chile.
Estela
E
Pérez
Emilio
E
Cardemil
D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D007930:Leucine