title: The strongly conserved lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer.
abstract: Lysine 256, a conserved amino acid of Saccharomycescerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis. These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography. The three variant enzymes showed almost unaltered Km for MnADP but about a 20 000-fold decrease in Vmax for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase. The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyruvate kinase-like activity. The dissociation constant for the enzyme-MnATP complex was 1.3 +/- 0.3 microM for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 microM, 17 +/- 2 microM, and 20 +/- 6 microM, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.
authors.affiliation: Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile.
authors.forename: H
authors.initials: H
authors.lastname: Krautwurst
authors.forename: S
authors.initials: S
authors.lastname: Bazaes
authors.forename: F D
authors.initials: FD
authors.lastname: González
authors.forename: A M
authors.initials: AM
authors.lastname: Jabalquinto
authors.forename: P A
authors.initials: PA
authors.lastname: Frey
authors.forename: E
authors.initials: E
authors.lastname: Cardemil
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D007700:Kinetics; D008239:Lysine / Q000032:analysis / Q000378:metabolism*; D008958:Models, Molecular; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D000255:Adenosine Triphosphate; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D008239:Lysine