Phosphatases active site

Kidney Bean Purple Acid Phosphatase Active Site... 

Interestingly, there is a close structural correspondence between the active sites of the haloperoxidases and the acid phosphatases that allows both peroxidase and phosphatase activity from the two types of enzymes [49-51], For instance, recombinant acid phosphatases from both Shigella flexneri and Salmonella enterica ser. typhimurium, when substituted by vanadate, are able to oxidize bromide when in the presence of hydrogen peroxide. However, the turnover rate is quite slow, which is in accord with the phosphatase active sites not being optimized for peroxidase activity [52],... 

Some properties of phosphopeptides make them preferable to the native phosphoprotein substrates for use with phosphate detection systems. The values of for peptide substrates are two to three orders of magnitude larger than for protein substrates and allow setting assays with an appropriate substrate concentration using standard phosphate detection approaches. In addition, short synthetic peptides are inexpensive and easy to obtain. Nonetheless, although phosphopeptide substrates are clearly useful in exploring interactions in the immediate vicinity of the phosphatase active site, they are unable to probe distant (allosteric) sites. 

Another important point from the HePTP case study illustrates epistemic challenges and their resolution with the use of small-molecule chemical probes. Although reversible oxidation of the phosphatase active site cysteines was hypothesized as one of the cellular pathways regulating protein phosphatase activity prior to our studies [40, 41], this mechanism was not experimentally demonstrated for HePTP. Identified small-molecule hits not only enabled biologically relevant inhibition of HePTP but also pointed... 

Figure 17-7 Two alternative mechanisms utilized by phosphatases to carry out hydrolysis of phosphate esters. The phosphoenzyme intermediate mechanism utilizes an amino acid (represented as -X] as a nucleophile to attack the phosphate ester, transferring the phosphoryi group and producing a short-lived phosphoenzyme intermediate. In the second step, water serves as the nucleophile, hydrolyzing the phosphoenyzme intermediate and regenerating the enzyme. This mechanism is used by the tyrosine phosphatases (nucleophile = cysteine) and E. coli alkaline phosphatase (active site nucleophile = Ser 102). The metallophosphatases do not proceed by formation of a phosphoenzyme intermediate but rather carry out hydrolysis by direct transfer of the phosphoryi group to a metal-coordinated water molecule.

Wang, L.K. Shuman, S. Mutational analysis defines the 5 -kinase and 3 -phosphatase active sites of T4 polynucleotide kinase. Nucleic Acids Res., 30, 1073-1080 (2002)... 

Recently, a somatic mutation of another PtdIns(3,4,5)P3-phosphatase, the 5-phosphatase SHIPl, was reported in primary myeloid leukemia cells (Luo et al, 2003). This mutation, in the phosphatase active site (V684E), generates a mutant catalytically defective in PtdIns(3,4,5)Pj 5-phosphatase activity. The consequences are enhanced Akt phosphorylation in response to interleukin-3, promotion of cell survival under conditions of serum deprivation, and resistance to apoptosis. Thus, mutations of the SHIPl gene may be involved in the development of acute leukemia and chemotherapy resistance through deregulation of the PtdIns(3,4,5)P3/Akt signalling pathway. 

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. 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

This is an emerging field that has not reached its final position yet. Members of the novel class of FCP, SCP, and HAD phosphatases require Mg2+ for catalysis. An aspartate residue within the active site signature motif (DxDx(T/V)) is essential to form an acyl-phosphate intermediate. Many members of the the HAD (haloacid dehalogenase) superfamily have phosphoesterase activity [3]. Some of those protein phosphatases act on... 

Figure 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

Renirie R, W Hemrika, R Wever (2000) Peroxidase and phosphatase activity of active-site mutants of vanadium chloroperoxidase from the fungus Curvularia inaequalis. J Biol Chem 275 11650-11657. 

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

Ser/Thr-protein phosphatases are ubiquitous enzymes which constitute the catalytic domains of multiprotein complexes. They are responsible for the dephosphorylation of a range of phosphoproteins. Several protein phosphatases have been characterized by X-ray crystallography and display an active site structure similar to purple acid phosphatase. 

While having three metal ions in an enzyme active site is uncommon, it is not unique to PLCBc. The well-known alkaline phosphatase from E. coli (APase) contains two zinc ions and a magnesium ion [67], whereas the a-toxin from Clostridiumperfringens [68]. and the PI nuclease from Penicillium citrinum [69] each contain three zinc ions. Indeed, the zinc ions and coordinating ligands of PI nuclease bear an uncanny resemblance to those of PLCBc as the only differ-... 

Intracellular and extracellular ROS activate tyrosine and serine-threonine kinases (i.e., the MAPK family members). Following TNF-a, TGF-f5 or EGF stimulation, intracellular ROS are generated which stimulate various signaling pathways [73], Tyrosine kinase receptors (e.g., EGF, PDGF and TGF-a) may be activated by ROS directly via protein sulfhydryl group modifications, or inhibition of phosphotyrosine phosphatases (PTPases) and subsequent receptor activation. The latter is possible as PTPases contain a redox-sensitive cysteine at their active site [78], and oxidation of protein sulfhydryl groups results in the inactivation of PTPases. 

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