title: Comparative steady-state fluorescence studies of cytosolic rat liver (GTP), Saccharomyces cerevisiae (ATP) and Escherichia coli (ATP) phospho enol pyruvate carboxykinases.
abstract: Two members of the ATP-dependent class of phospho enol pyruvate (PEP) carboxykinases (Saccharomyces cerevisiae and Escherichia coli PEP carboxykinase), and one member of the GTP-dependent class (the cytosolic rat liver enzyme) have been comparatively analyzed by taking advantage of their intrinsic fluorescence. The S. cerevisiae and the rat liver enzymes show intrinsic fluorescence with a maximum emission characteristic of moderately buried tryptophan residues, while the E. coli carboxykinase shows somewhat more average exposure for these fluorophores. The fluorescence of the three proteins was similarly quenched by the polar compound acrylamide, but differences were observed for the ionic quencher iodide. For the ATP-dependent enzymes, these last experiments indicate more exposure to the aqueous media of the tryptophan population of the E. coli than of the S. cerevisiae enzyme. The effect of nucleotides on the emission intensities and quenching efficiencies revealed substrate-induced conformational changes in the E. coli and cytosolic rat liver PEP carboxykinases. The addition of Mn2+ or of the adenosine nucleotides in the presence of Mg2+ induced an enhancement in the fluorescence of the E. coli enzyme. The addition of guanosine or inosine nucleotides to the rat liver enzyme quenched its fluorescence. From the ligand-induced fluorescence changes, dissociation constants of 40 +/- 6 microM, 10 +/- 0.8 microM, and 15 +/- 1 microM were obtained for Mn2+, MgATP and MgADP binding to the E. coli enzyme, respectively. For the cytosolic rat liver PEP carboxykinase, the respective values for GDP, IDP and ITP binding are 6 +/- 0.5 microM, 6.7 +/- 0.4 microM and 10.1 +/- 1.7 microM. A comparison of the dissociation constants obtained in this work with those reported for other PEP carboxykinases is presented.
authors.affiliation: Departamento de Química, Facultad de Ciencia, Universidad de Santiago de Chile.
authors.forename: M V
authors.initials: MV
authors.lastname: Encinas
authors.forename: M C
authors.initials: MC
authors.lastname: Rojas
authors.forename: H
authors.initials: H
authors.lastname: Goldie
authors.forename: E
authors.initials: E
authors.lastname: Cardemil
mesh_terms: D020106:Acrylamide; D000178:Acrylamides; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008345:Manganese; D009711:Nucleotides / Q000494:pharmacology; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012974:Sodium Iodide; D014364:Tryptophan / Q000032:analysis*
chemical_list: D000178:Acrylamides; D009711:Nucleotides; D020106:Acrylamide; D008345:Manganese; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D012974:Sodium Iodide