title: Functional evaluation of serine 252 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
abstract: Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase mutant Ser252Ala, affecting the conserved Walker A serine residue, was characterized to elucidate the role of this serine residue. The substitution did not result in changes in the protein structure, as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analysis of the mutated enzyme in both directions of the main reaction and in the two secondary reactions showed an approximately 50-fold increase in apparent K(m) for oxaloacetate with minor alterations in the other kinetic parameters. These results show that the hydroxyl group of serine 252 is required for proper oxaloacetate interaction.
authors.affiliation: Universidad de Santiago de Chile, Casilla, Santiago, Chile.
authors.forename: Daniel
authors.initials: D
authors.lastname: Castillo
authors.forename: Carolina
authors.initials: C
authors.lastname: Sepúlveda
authors.forename: Emilio
authors.initials: E
authors.lastname: Cardemil
authors.forename: Ana M
authors.initials: AM
authors.lastname: Jabalquinto
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D016384:Consensus Sequence; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010071:Oxaloacetates / Q000378:metabolism; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine / Q000032:analysis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D010071:Oxaloacetates; D011994:Recombinant Proteins; D012694:Serine; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D000409:Alanine