The recent studies demonstrated that 20 % of relapsed acute lymphoblastic leukemia are associated with mutations in NT5C2 encoding cytosolic purine 5'-nucleotidase (cN-II). The identified point mutations lead to a biosynthesis of hyperactive enzyme that inactivates drugs used for chemotherapy.
The aim of this project was detailed biochemical and structural characterization of representative hyperactive cN-II mutants. In this study, we analyzed the most common mutant R367Q. Kinetic analysis of nucleotidase reaction with inosine monophosphate as a substrate showed that the KM value of the R367Q is significantly decreased in the absence of physiological activator of the enzyme - ATP, i.e the KM value was determined to be 5.82 ± 1.69 mM compared to Km of 33.55 ± 9.14 mM for the wild-type cN-II. On the contrary, kinetic parameters of the R367Q mutant and wild-type enzyme were not different in the presence of ATP. The analysis of enzyme kinetics indicates that the mutant possessed abolished allosteric regulation being constitutively active.
Biophysical analysis showed that the R367Q cN-II as well as wild-type protein assembled properly into tetramer and thermal stability of the proteins was virtually identical exhibited melting temperature about 55 °C. Subtle changes in conformational properties were been also revealed by circular dichroism spectroscopy and tryptophan-based fluorimetry. The X-ray crystallography together with mass spectrometric studies demonstrated that the overall shape of the wild-type and mutant protein is indistingushable. Nevertheless the integration of the protein crystal structure with mass spectrometric data showed that all of the regions with altered topology are located at the oligomeric interface of the cN-II. Interestingly, the location of the perturbed regions is consistent with the distribution of other identified mutations causing the enzyme hyperactivity. It suggests that oligomeric interface of cN-II might be an important site for pharmacological intervention of relapsed ALL.