Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy

María Victoria Encinas, Fernando D. González-Nilo, Hughes Goldie, Emilio Cardemil

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the γ-phosphoryl group of ATP to yield PEP, ADP, and CO2. The interaction of the enzyme with the substrates originates important domain movements in the protein. In this work, the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phosphopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatized enzyme retained the substrate-binding characteristics of the native protein, allowing the determination of several protein-ligand dissociation constants, as well as the role of Mg2+ and Mn2+ in substrate binding. The binding affinity of PEP to the enzyme-Mn2+ complex was -8.9 kcal·mol-1, which is 3.2 kcal·mol-1 more favorable than in the complex with Mg2+. For the substrate nucleotide-metal complexes, similar binding affinities (-6.0 to -6.2 kcal·mol-1) were found for either metal ion. The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns. These lifetimes were markedly altered in the derivatized enzyme-PEP-Mn complexes, and smaller changes were obtained in the presence of other substrates. Molecular models of the P-pyridoxyl-E. coli PEP carboxykinase showed different degrees of solvent-exposed surfaces for the P-pyridoxyl group in the open (substrate-free) and closed (substrate-bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution.

Original languageEnglish
Pages (from-to)4960-4968
Number of pages9
JournalEuropean Journal of Biochemistry
Volume269
Issue number20
DOIs
Publication statusPublished - 2002

Keywords

  • Conformational changes
  • Escherichia coli phosphoenolpyruvate carboxy-kinase
  • Ligand binding
  • P-pyridoxyl fluorescence spectroscopy

ASJC Scopus subject areas

  • Biochemistry

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