Protein tyrosine phosphatases Active site

Studies of the oxidation of organic sulfides with amino acid-derived ligands in acetonitrile revealed very little difference between the mechanism of their oxidation and that of halides, except for one major exception. Despite the fact that acid conditions are still required for the catalytic cycle, hydroxide or an equivalent is not produced in the catalytic cycle, so no proton is consumed [48], As a consequence, there is no requirement for maintenance of acid levels during a catalyzed reaction. Peroxo complexes of vanadium are well known to be potent insulin-mimetic compounds [49,50], Their efficacy arises, at least in part, from an oxidative mechanism that enhances insulin receptor activity, and possibly the activity of other protein tyrosine kinases activity [51]. With peroxovanadates, this is an irreversible function. Apparently, there is no direct effect on the function of the kinase, but rather there is inhibition of protein tyrosine phosphatase activity. The phosphatase regulates kinase activity by dephosphorylating the kinase. Oxidation of an active site thiol in the phosphatase prevents this down-regulation of kinase activity. Presumably, this sulfide oxidation proceeds by the process outlined above. 

Chia JY, Gajewski JE, Xiao Y et al (2010) Unique biochemical properties of the protein tyrosine phosphatase activity of PTEN-demonstration of different active site structural requirements for phosphopepride and phospholipid phosphatase activities of PTEN. Biochim Biophys Acta 1804 1785-1795... 

Zhang, Z.-Y., Dixon, J. E. Active site labeling of the yersinia protein tyrosine phosphatase The determination of the pKa of active site cysteine and the function of the conserved histidine 402. Biochem. 32 (1993) 9340-9345. 

Nonreceptor protein tyrosine phosphatases are structurally different from serine-threonine phosphatases and contain a cysteine residue in their active sites. The... 

N-Nitrosamines have been shown to be inhibitors of cysteine-containing enzymes. For example, dephostatin and other N-methyl-N-nitrosoanilines (1) were found to be inhibitors of the protein tyrosin phosphatases, papain and caspase [90,91]. Inhibition results from the S-nitrosation of the critical cysteine residues in the active sites of the enzymes by the nitrosamines. Compounds 6 and 7 have been found to inhibit thrombus formation in arterioles and venules of rats [92], while N-nitrosamide 9 exhibited vasodilation and mutagenicity as a result of NO release [93]. 

The subcellular localization of protein tyrosine phosphatases is an important aspect of their function. The sequences of cytoplasmic protein tyrosine phosphatases frequently demonstrate sequence signals specifying a particular subcellular localization. This ensures that protein tyrosine phosphatases are only active at defined subcellular sites. The presence of SH2 domains in cytoplasmic protein tyrosine phosphatases also shows that these are coupled, via SH2-phosphotyrosine interactions, to specific substrates, where they then perform their actual function. 

The insulin receptor substrate IRS couples the insulin receptor to sequential effector molecules (review Ogawa et al., 1998). On binding of insulin to the insulin receptor, the tyrosine kinase activity of the receptor is stimulated. The IRS protein is phosphory-lated at several Tyr residues, which then serve as attachment points for sequential effector molecules as e.g. the Grb2-mSos complex, the P13-kinase and the protein tyrosine phosphatase SHP-2. The IRS protein also has a phosphotyrosine binding domain and a PH domain. Both modules are required for signal transduction in vivo. It is assumed that the PTB domain binds to autophosphorylation sites of the insulin receptor and that the PH domain is involved in membrane association of IRS. 

Many peroxovanadates have potent insulin-mimetic properties [1,2]. Apparently, this functionality derives from the ability of these compounds to rapidly oxidize the active site thiols found in the group of protein tyrosine phosphatases that are involved in regulating the insulin receptor function [3], The discovery of vanadium-dependent haloperoxidases in marine algae and terrestrial lichens provided an additional stimulus in research toward obtaining functional models of peroxidase activity, and there is great interest in duplicating the function of these enzymes (see Section 10.4.2). 

Liu S, Zhou B, Yang H et al (2008) Aryl vinyl sulfonates and sulfones as active site-directed and mechanism-based probes for protein tyrosine phosphatases. J Am Chem Soc 130 8251-8260... 

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). 

Fig. 7.2 Tlie crystal structure of mammalian Ser/Thr protein phosphatase-1, complexed with the toxin mycrocystin was determined at 2.1 A resolution. PPl has a single domain with a fold, distinct from that of the protein tyrosine phosphatases. The Ser/Thr protein phosphatase-1, is a metalloenzyme with two metal ions positioned at the active site with the help of a p-a-p-o-p scaffold. A dinuclear ion centre consisting of Mn2+ And Fe2+ g situated at the catalytic site that binds the phosphate moiety of the substrate. Ser/Thr phosphatases, PPl and PP2A, are inhibited by the membrane-permeable ocadaic acid and by cyclic hexapeptides, known as microcystins. The toxin molecule is depicted as a ball-and-stick structure. On the left and on the ri t, two different views of the same molecule are shown. Microcystin binds to three distinct regions of the phosphatase to the metaLbinding site, to a hydrophobic groove, and to the edge of a C-terminal groove in the vicinity of the active site. At the surface are binding sites for substrates and inhibitors. These ribbon models are reproduced vnth permission of the authors and Nature from ref. 9.

Site-specific incorporation of a nonhydrolyzable phosphotyrosine analog revealed a role for phosphorylation of protein tyrosine phosphatase SHP-2 in cell signaling (59). The phosphorylated SHP-2 protein showed improved activity in catalyzing phosphate release than its nonphosphorylated counterpart. 

Mechanistically, inhibition must not necessarily block the active site itself, but it can exert allosteric effects on the substrate-binding pocket, which thereby enhances or suppresses enzymatic activity. Additional considerations regarding enzymatic reactions are discussed in Reference 86. SAR by NMR has been successfully applied to various systems [i.e., for disrupting intracellular protein-protein binding (87) as well as cytokine-receptor interaction (88)]. High-affinity enzyme inhibitors have been developed by this technique [e.g., for the metalloproteinase Stromelysin (89) and the protein tyrosine phosphatase IB (90)]. 

Vanadium(IV) complexes with two dipeptides glycyl-tyrosine and glycyl-phenylalanine (Gly-Tyr and Gly-Phe, respectively) and their oxovanadium(lV) and (V) complexes have been observed.382, 01 The complex formed with Gly-Tyr showed coordination by the peptide backbone moieties, and no interaction from the distal phenolic hydroxy group.601 A histidine-tyrosine derivatized peptide was shown to complex Viv in a pentadentate manner (120).564 The adduct between VlV and a model of the active-site peptide of protein tyrosine phosphatases has been spectroscopically characterized.602 Similar characterization of in serum suggests that protein complexes form, and that both Viv and Vv can exist as complexes with transferrin and albumin.603 ESEEM has been used to characterize the complexes of apoferritin with V02+ and suggests that the carboxylates, one water molecule, and one histidine ligand fill the coordination sites of the vanadium.604... 

VI-IX are structurally characterised, vanadate-inhibited phosphatases. VI, Rat prostat acid phosphatase VII, bovine phosphotyrosyl phosphatase VIII, mammalian protein tyrosine phosphatase PTP-IB (mutant Cys215Ser) IX, E. coli alkaline phosphatase. For comparison, the active centre of vanadate-dependent haloperoxidases (VHPO) (V), is also shown. The structures Xa and Xb have been proposed, based on EPR, for the vanadyl complexes formed with the PTP-IB active site peptide Val-His-Cys-Ser-Ala-Gly. 

However the development of inhibitors for PTPs with suitable drug-like features of selectivity, absorption, distribution, metabolism, excretion and toxicity (ADMET) has proven to be challenging in part due to the highly conserved and charged active site [39]. A potent inhibitor of human low molecular weight protein tyrosine phosphatase is a 3-phenoxyphenyl derivative of an 5 -arylidene-2,4-thiazolidinedione [40]. 

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