The Metal Coordination of sCD39 during ATP Hydrolysis

When VO2+ was used as a substitute for Ca2+, the ATPase activity of soluble CD39 was 25% of that with Ca2+ as cofactor. Protein ligands of the VO2+-nucleotide complex bound to the catalytic site of soluble CD39 were characterized by electron paramagnetic resonance (EPR) spectroscopy. The EPR spectrum contained one species designated T with VO2+-AMPPNP as ligand. Two species D1 and D2 were observed when VO2+-AMPCP was bound to soluble CD39. The results suggest that species D1 and D2 represent the metal-ADP complexes at the catalytic site of soluble CD39 corresponding to the intermediate formed during ATP hydrolysis and the substrate for further hydrolysis, respectively.VO2+ can functionally substitute for Ca2+ as a cofactor of sCD39, and it produces four different EPR features when bound in the presence of different nucleotides or in the absence of nucleotide. The metal coordination for each conformation corresponding to each EPR species is proposed, and the mechanism of sCD39 catalysis is discussed.Ecto-Nucleoside triphosphate diphosphohydrolases (E-NTPDases, formerly called ecto-ATPases) hydrolyze nucleotides in the presence of divalent cations and are insensitive to inhibitors of P-type, F-type, and V-type ATPases [1]. Three isoforms that differ in the ratio of ATPase/ADPase activity are present on the cell surface [2]: E-NTPDase1 with a ratio of 1, E-NTPDase2 with a ratio of 10 and E-NTPDase3 with a ratio of 3–5. NTPDases are important in many physiological processes like cell motility, adhesion, nonsynaptic information transfer, secretion, regulation of hemostasis and ectokinases [1]. Understanding the enzymatic mechanisms of the NTPDases will help description of their physiological functions, and development of strategies to regulate the functions of the enzymes.The catalytic mechanism of NTPDases is not known even though some basic facts of the catalysis have been established. NTPDases do not form phosphorylated intermediates during catalysis, a conclusion als