Ligand interactions and protein conformational changes of phosphopyridoxyl‐labeled Escherichia coli phospho enol pyruvate carboxykinase determined by fluorescence spectroscopy

Escherichia coli phospho enol pyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the γ‐phosphoryl group of ATP to yield PEP, ADP, and CO 2 . 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 Mg 2+ and Mn 2+ in substrate binding. The binding affinity of PEP to the enzyme–Mn 2+ complex was −8.9 kcal·mol −1 , which is 3.2 kcal·mol −1 more favorable than in the complex with Mg 2+ . 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 degree s 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.