Phosphatases catalytic mechanism

A determination of the pH dependence of the lanthanum hydroxide gel-promoted hydrolysis of /3-glyceryl phosphate revealed that the two maxima exist in the pH-rate profile, one at pH 8.6 which presumably involves the species La (OH)+2 and another (smaller) maximum at pH 10.4 which involves the species La (OH) 2+ (4). Presumably the same kind of catalytic mechanism is operative in both cases. These reactions may serve as models for the metal ion-promoted alkaline phosphatases which have been shown to proceed with P—O cleavage (and with no oxygen exchange). 

Although the detailed catalytic mechanisms of these phosphatases have not been elucidated, an accepted general mechanism is that the two metal ions are cooperatively working by interacting directly with the scissible phosphate and stabilizing the pentacovalent intermediate (33, 45). Moreover, one zinc(II) ion generates the attacking OH ion. 

Some of the polymerases exist as single polypeptide chains, while others function only as large complexes. In every case a two-metal ion catalytic mechanism with in-line nucleotidyl transfer,269 illustrated in Fig. 27-13, appears to be used by the enzymes.267 270 Two-metal ion catalysis is also observed for phosphatases and ribozymes (Chapter 12). 

Denu, J.M. and J.E. Dixon. 1995. A catalytic mechanism for the dual-specific phosphatases. Biochemistry 92 5910-5914. 

On the other hand, a particular protein function can be realized with different protein folds, and an example of this are protein phosphatases. Protein phosphatases feature two distinctively different catalytic mechanisms for hydrolytically cleaving phosphorylated amino acid residues. The active sites of serine/threonine protein phosphatases (PPs) contain two metal centers that directly activate a water molecule for nucleophilic attack of the phosphate ester bond. In contrast, protein tyrosine phosphatases (PTPs) [105] possess a Cys residue present in the active site loop containing the conserved PTP signature motif HCXXXXXRS. The Cys sidechain acts as the attacking nucleophile in the formation of a phosphocysteine intermediate, which is eventually hydrolyzed by a water molecule [106], The same catalytic mechanism is also shared by dual-specificity phosphatases (see below). 

Figure4.1 Protein tyrosine phosphatase IB (2hnq (a)) and CDC25B (lcwt (b)) employ the same catalytic mechanism for hydrolysis of phosphorylated substrates but share no sequence homology and exhibit very different folds. The similarity of their binding sites can, however, be detected on the level of the interaction properties exposed to a ligand.

Figure 5 (a) Proposed catalytic mechanism of protein tyrosine phosphatases, (b) Structure of PTPl B (gray) with a phosphopeptide substrate (goid) showing the relative positions of catalytic residues to the phosphotyrosine moiety. 

Kolmodin K, Aqvist J. The catalytic mechanism of protein tyrosine phosphatases revisited. EEBS Lett. 2001 498 208-213. 

Figure 8 Kinase-catalyzed phosphorylation and phosphatases-catalyzed dephosphorylation reactions, (a) Catalytic mechanism of protein kinases (b) Catalytic mechanism of bimetallic pSer/pThr or dual specifity protein phosphatases (c) Catalytic mechanism of pTyr phosphatases.

Fig. 8 Catalytic mechanism of alkaline phosphatase reaction [44]. The initial alkaline phosphatase (E)-cataiyzed reaction consists of a substrate (DO-Pi) binding step, phosphate-moiety transfer to Ser-93 (in the TNAP sequence of its active site), and product alcohol (DOH) release. In the second part of the reaction, phosphate is released through hydrolysis of the covalent intermediate (E-Pi) and non-covaient compiex (E Pi) of inorganic phosphate in the active site. In the presence of alcohol molecules (AOH), phosphate is aiso reieased via a transphosphorylation reaction...

McCain DF, Catrina IE, Hengge AC et al (2002) The catalytic mechanism of Cdc25A phosphatase. J Biol Chem 277 11190—11200... 

We performed a series of experiments to elucidate the MOA of the novel TNAP inhibitors. The catalytic mechanism by which TNAP degrades PR consists of rapid phosphorylation of the active site in the presence of the phospho-donor substrate and a rate-limiting dephosphorylation by the phospho-acceptor substrate, either water or amino-containing alcohols (Fig. 5). All the TNAP inhibitors reported to date are uncompetitive with respect to phos-pho-donors and are likely to be non- or uncompetitive with diethanolamine (DBA). The latter conclusion is based on the fact that the majority of the alkaline phosphatase assays are performed in the presence of saturating concentrations of DBA or other phosphor-acceptors. 

In addition, the crystal structure of the histidine acid phosphatase from the pathogen Francisella tularensis tHAP) has been solved showing that the three-dimensional structure is similar to that of prostatic acid phosphatase [8]. Moreover, a mutation of the Asp261 (the equivalent to Asp258 in prostatic acid phosphatase) traps the substrate 3 -AMP (3 -adenosine monophosphate) which provides supporting evidence to the importance of this residue in the catalytic mechanism [8]. 

Zhang ZY, Wang Y, Dixon JE (1994) Dissecting the catalytic mechanism of protein-tyrosine phosphatases. Proc Natl Acad Sci US A9L1624-1627... 

The dependence of the activity of calcineurin on the redox state of the metal center highlights its importance for catalysis and provides clues to its function in that process. Site-directed mutagenesis studies of PPl, calcineurin, and bacteriophage X protein phosphatase have also provided insights regarding the roles of non-ligand active site residues. Furthermore, the contributions afforded by studies of synthetic model compounds which mimic features of metallophosphatase active sites provide important clues to possible catalytic mechanisms. Indeed many of these models exhibit impressive rate enhancements for phosphate and phosphonate ester hydrolysis [54-62]. In this section we discuss current models regarding the mechanism of phosphate ester hydrolysis by calcineurin and other metallophosphatases in consideration of these studies. 

Figure 14 Catalytic mechanisms proposed for the purple acid phosphatases (1) attack of a terminal hydroxide on the Fe on a monodentate phosphate ester substrate coordinated to the divalent metal site (2) attack of the bridging hydroxide on a bridging phosphate ester (3) attack of a hydroxide ion generated in the second coordination sphere of the Fe on a monodentate phosphate ester.

Figure 15 The three-metal-ion catalytic mechanism proposed for alkaline phosphatase (reproduced with...

Zhao, R., Collins, E.J., Bourret, R.B. and Silversmith, R.E. (2002). Structure and catalytic mechanism of the E. coli chemotaxis phosphatase CheZ. Nature Struct. Biol 9, 570-575. 

Box 3.1 Protein Tyrosine Phosphatases (PTPs) and Their Catalytic Mechanism... 

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