Abstract

Ecto-nucleoside triphosphate diphosphohydrolase 3 (eNTPDase-3, also known as HB6 and CD39L3) is a membrane-associated ecto-apyrase. Only a few functionally significant residues have been elucidated for this enzyme, as well as for the whole family of eNTPDase enzymes. Four highly conserved regions (apyrase conserved regions, ACRs) have been identified in all the members of eNTPDase family, suggesting their importance for biological activity. In an effort to identify those amino acids important for the catalytic activity of the eNTPDase family, as well as those residues mediating substrate specificity, 11 point mutations of 7 amino acid residues in ACR1-4 of eNTPDase-3 were constructed by site-directed mutagenesis. Mutagenesis of asparagine 191 to alanine (N191A), glutamine 226 to alanine (Q226A), and arginine 67 to glycine (R67G) resulted in an increase in the rates of hydrolysis of nucleoside diphosphates relative to triphosphates. Mutagenesis of arginine 146 to proline (R146P) essentially converted the eNTPDase-3 ecto-apyrase to an ecto-ATPase (eNTPDase-2), mainly by decreasing the hydrolysis rates for nucleoside diphosphates. The Q226A mutant exhibited a change in the divalent cation requirement for nucleotidase activity relative to the wild-type and the other mutants. Mutation of glutamate 182 to aspartate (E182D) or glutamine (E182Q), and mutation of serine 224 to alanine (S224A) completely abolished enzymatic activity. We conclude that the residues corresponding to eNTPDase-3 glutamate 182 in ACR3 and serine 224 in ACR4 are essential for the enzymatic activity of eNTPDases in general, and that arginine 67, arginine 146, asparagine 191, and glutamine 226 are important for determining substrate specificity for human ecto-nucleoside triphosphate diphosphohydrolase 3.