Study of some physical properties of gmp kinase from haemophilus influenzae

The proteins guanylate monophosphate kinases (GMPKs) are component parts of cytosolic NMP kinase family with a very important role in the activation of guanosine analog pro-drugs, used both in cancer and virosis therapies. GMPKs have been characterized as monomeric enzymes in prokaryote and oligomeric in eukaryote organisms. The primary structure of GMPK from Haemophilus influenzae contains one Trp and 10 Tyr residues, responsible for its fluorescence emission.The fluorescence spectrum of GMPK presents the Trp emission at λ_em = 320 nm. The change of protein fluorescence spectrum by the binding of 8-Anilino-1-napthalene sulfonic acid ammonium (ANSA), Guanosine monophosphate (GMP), and Adenosine diphosphate (ATP) ligands to GMPK was monitored. The ligand binding induces a static type quenching of protein fluorescence emission. The affinity constants show that ANSA and GMP are more efficient than ATP in the binding process (K_ANSA = 4.5×10⁴ M^-1, K_GMP = 3.3×10⁴ M^-1, K_ATP = 1.56×10² M^-1). The influence of temperature on the GMPK fluorescence emission was also monitored. The preliminary studies by differential scanning calorimetry (DSC) on GMPK confirm the value of the denaturation temperature, previously deduced from the fluorimetric experiments. Thus, the denaturation of the protein, monitored by DSC, is taking place on a narrow temperature interval, around the value of 50 °C.