Phosphatases are a large group of enzymes found in many different enzyme superfamilies that catalyze phosphoryl transfer reactions. In general, phosphatases can be arranged according to their substrates specificities, as exemplified by tyrosine specific phosphatases, serine/threonine specific phosphatases, dual specificity phosphatases, histidine phosphatases and lipid phosphatases. Owing to the response of their catalytic activity to the pH of the environment, these enzymes have been further classified as alkaline phosphatases, which are most active in an alkaline media, and acid phosphatases, which display higher activities under acidic reaction conditions.
Although many different types of phosphatases are known, with only a few exceptions they all rely on nucleophilic catalysis to promote phosphoryl transfer reactions. In 1961, Engstrom discovered that catalysis of phosphate ester hydrolysis by an Escherichia coli serine alkaline phosphatase proceeds through a pathway in which a phosphoseryl intermediate is generated. Later work carried out by Lipmann, in which the serine phosphate intermediate was isolated after incubating alkaline phosphatase with inorganic phosphate, confirmed the earlier finding by Engstrom. The results of experiments using a mixture of P32-phosphate and P31-glucose 6-phosphate enabled Lipmann to prove that a phosphoseryl intermediate is formed in the active site of the phosphatase.
Important insight into the mechanisms of phosphatase catalyzed reactions has come from studies probing the phosphorus stereochemical outcomes of the processes. Specifically, phosphatase catalyzed hydrolysis reactions of phosphate monoesters were shown to take place with retention rather than inversion of P-stereochemistry, an observation that demonstrates that the processes occur through two step routes, each of which takes place with inversion of P-stereochemistry. In the case of the serine phosphatase discussed above, the first mechanistic step involves substitution at phosphorus by the serine-102 hydroxyl group displacing the alcohol leaving group of the phosphate mono ester substrate. The phosphoenzyme intermediate generated in this step then undergoes a displacement reaction, in which water or an alcohol serves as the nucleophile, to produce product and the regenerated active enzyme.
As mentioned above, phosphoserine, phosphohistidine and phosphoaspartate intermediates have been identified as participants in the chemical mechanisms of phosphatase catalyzed phosphoryl transfer reactions. In addition, a cysteine phosphate has been shown to serve as an intermediate in the hydrolysis reaction catalyzed by protein-tyrosine-phosphatase. An interesting feature of this process is that at least three distinctly different phosphoenzyme intermediates are formed in the reaction pathway.
Additional information leading to a more detailed understanding of how phosphatases catalyze reactions has come from studies exploring the nature of the phosphoryl transfer process. The two limiting mechanisms that are typically considered operable for nonenzymatic solution phosphoryl-transfer reactions involve either a dissociative or an associative process. In the transition state for the dissociative mechanism, the bond between phosphorus and the leaving group is broken either extensively or completely and bond formation to the incoming nucleophile is either not well advanced or absent. In contrast, in the associative pathway the respective bonds between the incoming nucleophile and phosphorus and the departing leaving group and phosphorus are formed and broken in a concerted (not necessarily synchronous) manner.
The dissociative and associative reaction pathways for tyrosine phosphatase promoted reactions have been studied by using density functional theory calculations, which yielded energy barriers and geometries for points along each reaction pathway. The energy barrier of the dissociative process with early proton transfer to the leaving group was predicted to be 9 kcal/mol, while that for the associative reaction with late proton transfer was predicted to be 22 kcal/mol. These results are in accord with experimental evidence that showed that tyrosine phosphatase catalyzes phosphate ester hydrolysis of a dianionic substrate via a dissociative pathway.
Phosphatase Members of the Haloacid Dehalogenase Enzyme Superfamily (HADSF)
The Haloacid Dehalogenase (HAD) superfamily, named after the first family member to be structurally characterized, is a ubiquitous family of enzymes. Presently, ca. 50,000 deposited gene sequences have been identified that encode proteins in this superfamily. The HAD superfamily is highly evolvable and, thus, it is ideal to employ in formulating and testing theories about enzyme superfamily nucleation and growth.
A large subgroup of HAD superfamily (HADSF) members serve as phosphatases. All enzymes in this family possess a highly conserved core domain that contains amino acid side chains that participate in catalysis of phosphoryl group transfer processes. In addition, all members possess an Asp nucleophile residue in their active sites, which participates as a nucleophile in catalyzing the phosphoryl transfer reactions, and all members of the HADSF, except haloalkanoic acid dehalogenases, which catalyze a carbon group transfer reactions, utilize Mg2+ as a cofactor for catalysis. The catalytic scaffold of the core domain of phosphatase members of the HADSF is formed by 4-loops that position both the "core catalytic residues" and "diversification residues". The core residues include a loop 1 Asp that serves as a nucleophile and a loop 4 Asp that participates as a general acid/base and to bind Mg2+ ion. Additional catalytic residues in the active sites of HADSF members include loop2 Ser/Thr and loop 3 Arg/Lys side chains that serve to position the phosphate moiety of the substrate via hydrogen bonding interactions.
Many HADSF members possess cap domains in addition to core domains. Typically, the cap domains of these enzymes contain amino acid side chains that participate in binding to the leaving groups of phosphoryl transfer reactions and, as a result, contribute to substrate recognition. In addition, the cap domains play a role in separating solvent and substrate by functioning as a hinged “lid” over the substrate-filled active site of the core domain. The caps are connected at different sites of the enzyme backbone, a phenomenon that serves as the basis for subclassification of members of this family. These HAD subclasses are referred to as C0 caps, which have only small inserts in either of the two points of cap insertion, C1 caps, whose caps are defined as inserts occurring in the middle of a E-hairpin of the flap motif, and fold and C2 caps, which have inserts occurring in the linker positioned immediately after loop 2.