Although NPP primarily catalyzes phosphodiester hydrolysis, the enzyme will also catalyze the hydrolysis of phosphate monoesters, though to a much smaller extent. NPP preferentially hydrolyzes phosphate diesters over monoesters by factors of 10-10, depending on the identity of the diester substrate. This ability to catalyze a reaction with a secondary substrate is known as enzyme promiscuity, and may have played a role in NPP's evolutionary history.
NPP's promiscuity enables the enzyme to share substrates with alkaline phosphatase (AP), another member of the alkaline phosphate superfamily. Alkaline phosphatase primarily hydrolyzes phosphate monoester bonds, but it shows some promiscuity towards hydrolyzing phosphate diester bonds, making it a sort of opposite to NPP. The active sites of these two enzymes show marked similarities, namely in the presence of nearly superimposable Zn bimetallo catalytic centers. In addition to the bimetallo core, AP also has an Mg ion in its active site.