Fluorescent study of the interaction between Staphylococcus aureus UMP kinase and UTP

Fluorescence-based techniques applied to the proteins are giving information on their binding sites for ligands, interactions with solvent, the degrees of flexibility, internal motions, rotational diffusion coefficients, etc. The fluorescence behaviour of one member of NMP kinase family, namely, Staphylococcus aureus UMP kinase, was tested. NMP kinases are ubiquitous enzymes of the living cells, very important in the cellular energetic metabolism, and also in synthesis of nucleic acid precursors. It seems that Staphylococcus aureus UMP kinase has a different behaviour, as compared to UMP kinases from Streptococcus pneumoniae or from Escherichia coli. The binding of a specific ligand, UTP, to this UMP kinase seems to be a cooperative process, being accompanied by about 8-fold decrease in the intrinsic fluorescence, in UTP concentration range (50-1,500) μM. UMP kinase from Staphylococcus aureus is exhibiting an intrinsic fluorescence, due to its unique tryptophan, decreasing with the increase of UTP concentration. In the hypothesis of a collisional quenching of fluorescence, the fluorescence data were fitted by the Stern-Volmer equation. A very large Stern-Volmer constant, K D , of 5 × 10 3 M -1 was obtained. Using a mean lifetime fluorescence of tryptophan, in neutral aqueous solutions, we found a bimolecular quenching constant, kq, of 2 × 10 12 M -1 s -1 . This value is larger than that predicted for a diffusion-controlled reaction, therefore implying a complex formation between the enzyme and the ligand.