Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: physicochemical characteristics of the nucleotide binding site, as deduced from fluorescent spectroscopy measurements.
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is inactivated by the fluorescent sulfhydryl reagent N-(iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS). The inactivation reaction follows pseudo-first-order kinetics with respect to active enzyme to less than 10% remaining enzyme activity, with a second-order inactivation rate constant of 2.6 min-1 mM-1 at pH 7.5 and 30 degrees C. A stoichiometry of 1.05 mol of reagent incorporated per mole of enzyme subunit was found for the completely inactivated enzyme. Almost complete protection of the enzyme activity and of dansyl label incorporation are afforded by MnADP or MnATP, thus suggesting that 1,5-IAEDANS interacts with an enzyme sulfhydryl group at the nucleotide binding site. The fluorescence decay of the AEDANS attached to the protein shows a single-exponential behavior with a lifetime of 18 ns. A comparison of the fluorescence band position and the fluorescence decay with those of the adduct AEDANS-acetylcysteine indicates a reduced polarity for the microenvironment of the substrate binding site. The quenching of the AEDANS moiety in the protein can be described in terms of a collisional and a static component. The rate constant for the collisional component is much lower than that obtained for the adduct in a medium of reduced polarity. These last results indicate that the AEDANS moiety is considerably shielded from the solvent when it is covalently attached to PEPCK.
Encinas
M V
MV
Departamento de Química, Facultad de Ciencia, Universidad de Santiago de Chile.
Quiñones
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V
Cardemil
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E
D001665:Binding Sites; D005456:Fluorescent Dyes; D007700:Kinetics; D009282:Naphthalenesulfonates / Q000494:pharmacology; D009711:Nucleotides; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D009711:Nucleotides; C006421:1,5-I-AEDANS; D010729:Phosphoenolpyruvate Carboxykinase (GTP)