Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases

Diadenosine polyphosphates (Ap(n)As) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface-located enzymes in an asymmetric manner, generating AMP and Ap(n-1) from Ap(n)A. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto-nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates diadenosine 5',5'''-P-1,P-3-triphosphate (Ap(3)A), diadenosine 5',5'''-P-1,P-4-tetraphosphate (Ap(4)A), and diadenosine 5',5'''-P-1,P-5-pentaphosphate, (Ap(5)A), and the diguanosine polyphosphate, diguanosine 5',5'''-P-1,P-4-tetraphosphate (Gp(4)G). Each of the three enzymes hydrolyzed Ap(3)A, Ap(4)A, and Ap(5)A at comparable rates. Gp(4)G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap(4)A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the alpha,beta-pyrophosphate bond. Ap(n)A hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (K-m) values for Ap(3)A were 5.1 mum, 8.0 mum, and 49.5 mum for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues.