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Nucleotide Pyrophosphatase/Phosphodiesterase (NPP) is a class of dimeric enzymes that catalyze the hydrolysis of phosphate diester bonds. NPP belongs to the Alkaline Phosphatase (AP) superfamily of enzymes. The NPP family of enzymes includes seven isoforms, some of which prefer nucleotide substrates, some of which prefer phospholipid substrates, and others of which prefer substrates that have not yet been determined. In eukaryotes, NPP enzymes are located in the cell membrane and hydrolyze extracellular phosphate diesters to affect a wide variety of biological processes.

Structure
The catalytic site of NPP consists of a two-metal-ion (bimetallo) Zn2+ catalytic core. These Zn2+ catalytic components are thought to stabilize the transition state of the NPP phosphoryl transfer reaction.



Mechanism
NPP catalyses the nucleophilic substitution of one ester bond on a phosphodiester substrate. The mechanism by which this phosphoryl transfer reaction is catalyzed is an area of ongoing research, though several extreme possibilities exist. The first mechanistic possibility is a dissociative (elimination-addition) mechanism, in which the phosphoryl transfer would involve a metaphosphate intermediate. The transition state for this transfer is referred to as “loose” in the scientific literature. The second mechanistic possibility is a concerted mechanism, which is an SN2 type of mechanism in which one bond is broken while another is simultaneous formed. The transition state of this SN2 type mechanism is referred to as “synchronous.” The final mechanistic possibility for phosphoryl transfer is an associative (addition-elimination) mechanism, in which a pentavalent phosphorane intermediate is formed. Transition states for this type of mechanism are referred to as “tight.” These three cases represent archetypes for the reaction mechanism, and the actual mechanism could fall somewhere in between.

Promiscuity
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 102-106, depending on the identity of the diester substrate. This ability to catalyze a reaction with a secondary substrate is known as enzyme promiscuity. This characteristic means that NPP is able to share substrates with alkaline phosphatase (AP), another member of the Alkaline Phosphate superfamily. Alkaline phosphatase primarily hydrolyzes phosphate monoester bonds, but interestingly 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 Zn2+ bimetallo catalytic centers. In addition to the bimetallo core, AP also has an Mg2+ ion in its active site.

Evolution
NPP belongs to the Alkaline Phosphatase superfamily, which is a group of evolutionarily related enzymes that catalyze phosphoryl and sulfuryl transfer reactions. This group includes phosphomonoesterases, phosphodiesterases, phosphoglycerate mutases, phophophenomutases, and sulfatases.