Vanadate-inhibited phosphatases

The pseudohalide azide inhibits VCIPO. The first stmcturally characterised VHPO had in fact been crystallised in its azide-inhibited form. Inhibition has also been noted with hydroxylamine and hydrazine. Further, structural analogues of vanadate, such as [ALF4] and phosphate, are potent inhibitors. In turn, vanadate inhibits many phosphatases (and other phosphate-metabolising enzymes). On the other hand, apo-VHPOs can exhibit some phosphatase activity, and vanadate-inhibited phosphatases show some haloperoxidase activity. These phenomena will be discussed in Section 5.2.1. 

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. 

Haloperoxidase Activity of Vanadate-inhibited Phosphatases Phosphatase Activity of Apo-haloperoxidases... 

Vanadate and Phosphatases. The structural similarity between vanadate and phosphate is impressively demonstrated by the fact that apo-VHPOs can exhibit some phosphatase activity, whereas certain vanadate-inhibited phosphatases exert haloperoxidase activity, a fact which roots in homologies of the active site protein pockets of both classes of enzymes, and the structural analogy of the active centers (the histidine-coordinated vanadate) in the VHPOs and the phosphatases (17) cf Fig. 2. Vanadate-inhibited phosphatases for which peroxidase activity has been reported are of bacterial Shigella flexneri, Salmonella enterica (18)) and fungal origin (ph3rtases from Aspergillus (19)). 

Abbreviations. a-M, a-mannosidase AP, acid phosphatase as-ni-ATPase, anion-stimulated, nitrate-inhibitable ATPase CCR, NAD(P)H-dependent cytochrome oreduc-tase cs-vi-ATPase, cation-stimulated, vanadate-inhibitable ATPase, CAT, catalase GS 1/11, glucan synthase 1 or 11 IDPase, inosine diphosphatase cs-PPase, cation-stimulated pyrophosphatase RNA polymerase, DNA-dependent RNA polymerase TP-25, 25 kDa tonoplast integral protein. 

RIPA lysis buffer (without vanadate and sodium fluoride inhibitor as they inhibit phosphatase activity)... 

Cuncic, C., N. Detich, D. Ethier, A.S. Tracey, M.J. Gresser, and C. Ramachandran. 1999. Vanadate inhibition of protein tyrosine phosphatases in Jurkat cells Modulation by redox state. J. Biol. Inorg. Chem. 4 354—359. 

The inhibition of PTPs by vanadate is of particular interest since this mode of action is supposed to be the primary effect exerted by vanadium applied as an insulin-mimetic agent (see 5.1.1.5). The vanadate-inhibited PTP-IB VIII in Figure 5.19, the structure of which was revealed both by single-crystal X-ray diffraction and two-dimensional H- N NMR spectroscopy, was prepared by treating the phosphatase with either vanadyl sulfate or bis(maltolato)oxovanadium(lV) (BMOV) (7a in Figure 5.2). Irrespective of the nature of the intrinsically applied vanadium species, the same compound with incorporated vanadate(V) was obtained, nicely demonstrating that the active species is vanadate(V), formed by elimination of the ligands and oxidation of to V. In vivo studies further showed that intracellular PTP-IB from rat heart tissue was actively inhibited, and autophosphorylation of the insulin receptor concomitantly enhanced. 

Fig. 2. Left The active centers of the bromoperoxidase (VBrPO) from A nodosum (6). Center The active center of the chloroperoxidase (VClPO) from C. inaequalis (8). Right Vanadate-inhibited rat prostate acid phosphatase. Some of the hydrogen-bonding interactions with amino acid side-chains are shown. For highlighted amino acids, see the text.

Vanadate (sodium orthovanadate or peroxovanadate) exhibits insulin-like effects in vitro (activation of insulin receptor tyrosine kinase, PI 3-kinase, Akt) and in vivo (diabetic rats, humans). These effects can be explained at least in part by the inhibition of phosphotyrosine phosphatases which deactivate the INSR tyrosine kinase. 

Hydrogen peroxide (H202) exhibits insulin-like activity in isolated cells. Like that of vanadate, this effect is thought to be mediated by inhibition of protein-tyrosine phosphatases. 

It is not clear whether V(V) or V(IV) (or both) is the active insulin-mimetic redox state of vanadium. In the body, endogenous reducing agents such as glutathione and ascorbic acid may inhibit the oxidation of V(IV). The mechanism of action of insulin mimetics is unclear. Insulin receptors are membrane-spanning tyrosine-specific protein kinases activated by insulin on the extracellular side to catalyze intracellular protein tyrosine phosphorylation. Vanadates can act as phosphate analogs, and there is evidence for potent inhibition of phosphotyrosine phosphatases (526). Peroxovanadate complexes, for example, can induce autophosphorylation at tyrosine residues and inhibit the insulin-receptor-associated phosphotyrosine phosphatase, and these in turn activate insulin-receptor kinase. 

As noted earlier, studies with inhibitors have been of great value. One mole of ouabain binds per enzyme complex and inhibits all enzyme functions. It provides a convenient marker for the extracellular surface of the enzyme. Oligomycin inhibits the (Na+, K+)-ATPase but not the K+-phosphatase reaction. It stimulates the ADP/ATP exchange reaction and this led to the postulate for two phosphoenzymes in the reaction scheme. Anomalous kinetic behaviour for (Na+, K+)-ATPase, over some years, was eventually recognized57 to be due to a vanadate impurity in ATP, which binds with high affinity to the low affinity ATP site and with low affinity to the high affinity ATP site. In accord with this, vanadate effectively inhibits the K+-phosphatase... 

Acid and alkaline phosphatases with phosphorylated intermediates are inhibited by vanadate. This has been exploited in the study of the role of alkaline phosphatase in mineralization.1069 Vanadate also inhibits the ATP-dependent degradation of proteins in reticulocytes.1070... 

Vanadate is reduced in red cells to vanadyl ion by intracellular glutathione following uptake through a phosphate transport system.1072,1073 Free vanadyl is normally unstable with respect to oxidation, but appears to be stable when complexed with intracellular proteins or smaller molecules.1074 Vanadyl ion is a relatively powerful inhibitor of (Na+, K+)-ATPase. For pure fractions of the enzyme, inhibition was nearly complete at less than 5 pmol dm-3 vanadyl ion.1075 The state of vanadyl ion at pH 7 is somewhat uncertain, but may involve a hydroxylated species. Vanadyl ion also inhibits alkaline phosphatase more effectively than does vanadate.1076... 

Extrapolating from well-characterized enzymatic inhibition in test tubes, numerous mechanistic ideas concerning the in vivo effects of vanadium compounds have been advanced. The effects of vanadium compounds as transition-state analogs of certain enzymes with a phosphoprotein intermediate in their reaction scheme is proposed to account for the action of vanadium [11] in many biological systems. Unfortunately, it is often difficult to determine if the inhibition observed in the test tube occurs in vivo. For example, although vanadate is a potent inhibitor of plasma membrane ion pumps (such as the sodium potassium ATPase) in the test tube, it is difficult to determine if these pumps are actually inhibited in animals exposed to vanadium compounds. Currently, the role of vanadium compounds as protein phosphatase (PTP) inhibitors is believed to be related to the metabolic effects of this... 

The active sites of these enzymes can have a nitrogen ligand, usually as histidine (acid phosphatases and some protein phosphatases), a nucleophilic serine residue (alkaline phosphatases), a cysteine residue in which the thiol group can form a covalent species with the phosphate ester (protein phosphatases), or an aspartate-linked phosphate (plasma membrane ion pumps). The inhibitory form of vanadium is usually anionic vanadate V(V), but cationic vanadyl V(IV) has also shown strong inhibition of some types of phosphorylase reactions. Above neutral pH, speciation of vanadyl ions produces anionic V(IV) species capable of inhibition of enzymes in the traditional transition-state analogue manner [5],... 

Swarup, G., S. Cohen, and D.L. Garbers. 1982. Inhibition of membrane phosphoty-rosyl-protein phosphatase activity by vanadate. Biochem. Biophys. Res. Commun. 107 1104-1109. 

Huyer, G. 1997. Mechanism of inhibition of protein tyrosine phosphatases by vanadate and pervanadate. J. Biolog. Chem. 272 843-851. 

The phosphorylated receptor appears to be capable of being dephosphorylated by the action of endogenous cytosolic phosphatases, as incubation of cells with vanadate, which inhibits the action of tyrosine-phosphate-specific phosphatases, increased the phosphorylation state of the receptor [60,61]. The significance of this observation has yet to be ascertained. 

Acid and alkaline phosphatases which are believed to pass through a phosphorylated intermediate during the hydrolysis of p-nitrophenolphosphate are inhibited by vanadate )68-69. In the case of alkaline phosphatase from E. Coli, V02+ inhibits better than vanadate(V) (Table 2)68. Typical inhibitor plots are shown in Fig. 10. The reduction of the effectiveness of VOz+ as an inhibitor by about a factor of five in tris buffer probably stems from the complexation of the metal ion by tris. Such complexation is known to occur21. ... 

Many polyoxometalate salts have been shown to be biologically active. Snch activity inclndes a highly selective inhibition of enzyme Ihnction (phosphatases, dehydrogenases, isomerases), especially with the smaller vanadate and molybdate oligomers (V4, M04, M05). In some cases, the selectivity seems to depend npon shape. 

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