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Abstract
The cytosolic 5’-nucleotidase II is an ubiquitous enzyme that belongs to the family of enzymatic proteins involved in the metabolism of nucleotides. Each of this proteins have a specific subcellular localization, substrate specificity and regulation mechanism. The cN-II is a soluble form most expressed in tissues with active DNA synthesis and rapid turnover of nucleic acids and precursors; it catalyzes preferentially (d)IMP and (d)GMP. The enzyme belongs to the HAD (Haloacid Dehalogenase) superfamily and it is a bifunctional enzyme: the mechanism of action involves the formation of a phosphorylated intermediate, which may be hydrolyzed (nucleotidase activity) or the phosphate may be transferred to a suitable acceptor (phosphotransferase activity). The enzymatic activity is regulated in a very complex manner by 2,3-BPG, ATP, ADP and diadenosine polyphosphates (in particular Ap4A), while inorganic phosphate (Pi) is the only known inhibitor. The enzymatic activity is Mg2+-dependent.
The ability of the cytosolic 5’-nucleotidase II to phosphorylate purine nucleosides and their analogs appears to be very important in the intracellular metabolism of these compounds:
the enzyme has been demonstrated to be responsible for the phosphorylation of antiviral and anticancer prodrugs. Moreover, the enzyme has been also shown to use several monophosphate analogues as substrate of the nucleotidase reaction, and resistance both in vitro and in vivo correlates with increased cN-II levels. The study of cN-II activity and
regulation could so be helpful to counteract the cN-II-related resistance to nucleoside analogs, or to develop new drugs specifically activated by cN-II.
The cytosolic 5’-nucleotidase II is an omotetramer composed by two identical dimers.
Each subunit contains the active site and two effector sites (effector site 1 and 2) that bind regulatory molecules affecting enzymatic activity. The position of these sites in the tridimensional structure of the protein has been identified by crystallographic structure studies. According to the results of the crystallographic studies, we could hypothesize that the effector site 1 (near subunit interface A) is the binding site of Ap4A or 2,3-BPG: these interaction efficiently glue the tetramer subunits together in pairs and suggest the modulation of subunit association to be the base of the activation induced by effectors at effector site 1. The effector site 2 could be the binding site for an adenylic nucleotide with regulatory function.
In this study the kinetic characteristics of two point mutants (M436W and F127E) in the effector site 2 is presented. The two amminoacidic residues has been mutated using a PCR-
Abstract _________________________________________________________________________
based site-directed mutagenic method on the bovine cN-II gene present in the pET-28c vector. The recombinant purified proteins have been characterized using a radiochemical assay to evaluate the effect of the point mutations on the active site and on the interaction with the effector molecules. Both mutations don’t affect the active site: the Km values for IMP (substrate for the nucleotidase reaction) and inosine (substrate for the phosphotransferase reaction) are in fact similar to the values of the wild type enzyme. The K50 value for Mg2+ is comparable to that of the wild type protein, as a confirm that mutations in the effector site 2 don’t affect the active site. The mutant M436W shows a 10- fold increase in the K50 value for Ap4A, and the activatory effect is 3-fold higher compared to the wild type enzyme. Both mutants don’t show any difference in the K50 values for the effectors ADP and ATP, but in the latter case the K50 ATP shows a sigmoid trend for both mutants. This suggests a cooperative binding of ATP to both the mutated enzymes.
These results allowed us to hypothesize that the effector site 2 is the binding site for ATP.
The variation of K50 value for Ap4A observed for the M436W mutant suggests that the structural analogy between ATP and Ap4A allows the latter to bind in the ATP-specific effector site, which could recognize only the phosphate moiety of the effector molecule.
