Phosphatases reactions

The use of QM-MD as opposed to QM-MM minimization techniques is computationally intensive and thus precluded the use of an ab initio or density functional method for the quantum region. This study was performed with an AMi Hamiltonian, and the first step of the dephosphorylation reaction was studied (see Fig. 4). Because of the important role that phosphorus has in biological systems [62], phosphatase reactions have been studied extensively [63]. From experimental data it is believed that Cys-i2 and Asp-i29 residues are involved in the first step of the dephosphorylation reaction of BPTP [64,65]. Alaliambra et al. [30] included the side chains of the phosphorylated tyrosine, Cys-i2, and Asp-i 29 in the quantum region, with link atoms used at the quantum/classical boundaries. In this study the protein was not truncated and was surrounded with a 24 A radius sphere of water molecules. Stochastic boundary methods were applied [66]. 

Figure 6.47 Two phosphatase reactions to overcome kinase reactions...

Figure 11.21 Outline of synthesis of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine. Note in the synthesis of phosphatidylinositol, the free base, inositol, is used directly. Inositol is produced in the phosphatase reactions that hydrolyse and inactivate the messenger molecule, inositol trisphosphate (IP3). This pathway recycles inositol, so that it is unlikely to be limiting for the formation of phosphatidylinositol bisphosphate (PIP )- This is important since inhibition of recycling is used to treat bipolar disease (mania) (Chapter 12, Figure 12.9). Full details of the pathway are presented in Appendix 11.5. Inositol, along with choline, is classified as a possible vitamin (Table 15.3).

Li+ may bind at the Na+ site with activation42 while K+ binds with inhibition. For these internal Na+ sites, Km (Na+) = 1.23 mmol dm 3 and KT (K+) = 4.5 mmol dm-3. Both high and low affinity sites exist for K+ for the high affinity sites Km (K+) = 0.08 mmol dm 3 and (Na+) = 14.3 mmol dm 3, thus showing good discrimination between K+ and Na+. The low affinity sites have Km (K+) = 1.2 mmol dm-3 and appear to be involved in the phosphatase reaction Na+ binds with inhibition [X, (Na+) = 4.5 mmol dm-3]. Other Group IA cations activate at the K+ sites thus there is evidence for two sites for Rb+ per ouabain site. 

For the ATPase activity of the enzyme, Mg2+ is required. Kinetic studies confirm that MgATP is the substrate and that while free ATP binds to the enzyme it inhibits activity. It appears that MgATP binds at high and low affinity sites, with Km values about 3 p,mol dm-3 and 0.2 mmol dm-3 respectively. For phosphorylation of the enzyme, saturation of the high affinity site is sufficient for maximum activity. Binding at the low affinity sites inhibits ADP/ATP exchange and may represent a modulator role for these sites. This low affinity site may be the site for the substrate in the K+-phosphatase reaction. 

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... 

It was shown previously (24) that KD0-8-phosphate was a weak end-product inhibitor of the synthase reaction. Both of the end products of this reaction, KDO and inorganic phosphate are weak mixed-function inhibitors of KD0-8-phosphate phosphatase. The reduced form of KD0-8-phosphate (the C-2 carbonyl was reduced to the corresponding diasterioisomeric alcohol with NaBH, Table V, 3 red.), an open chain analogue, was neither an inhibitor of KD0-8-phosphate synthase nor was it a substrate or inhibitor of the phosphatase reaction. These findings indicate that the mechanism of KD0-8-phosphate synthase does not involve the formation of a linear intermediate and that the KD0-8-phosphate phosphatase requires the phosphorylated substrate in the ring form rather than the linear form. 

Gluconeogenesis uses seven of the reactions in glycolysis, but three are replaced by the sum of the pyruvate carboxylase and phosphoeno/pyruvate carboxykinase reactions, the fructose 1,6-biphosphatase reaction, and the glucose 6-phosphatase reaction. Tables 4.7, 4.8, and 4.9 give the thermodynamic properties of these reactions and the net reaction for gluconeogenesis. 

Dihydroxyacetone phosphate may be reduced to glycerol-3-phosphate, and the latter can yield glycerol by a phosphatase reaction ... 

In combination with an alkaline phosphatase reaction-linked assay, these two schemes have been used successfully for the identification of mutations in the rpoB gene of Mycobacterium tuberculosis from clinical isolates that show rifampin resistance (Rifr). The advantages and disadvantages of the new approach are discussed. 

The sensitivity of ELISA can be further increased by coupling to a second enzyme reaction. For example, the alkaline phosphatase reaction can be used to convert NADP+ into NAD+, which in the presence of a dehydrogenase can be used to convert a leuco-dye into coloured product. 

Computational enzymology Protein tyrosine phosphatase reactions... 

In the indirect method, crystalline phosphorylase a is incubated with the enzyme preparation to be assayed, aliquots are withdrawn, diluted (which stops the phosphatase reaction), and the diluted solutions are assayed in the absence of 5 -AMP for remaining phosphorylase a activity by incubation with glucose-l-phosphate and glycogen and determining the inorganic phosphate released. 

Competitive inhibition can occur in freely reversible reactions owing to accumulation of products. Even in reactions that are not readily reversible, the product can function as an inhibitor. In the alkaline phosphatase reaction, in which hydrolysis of a wide variety of organic monophosphate esters into the corresponding alcohols (or phenols) and inorganic phosphates occurs, the inorganic phosphate acts... 

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...

Fig. 4 Mechanism of action (MOA) and inhibition studies of ML119 (compound 1) with HePTP and HePTP mutants, (a) Progress curves of HePTP (6.25 nM) activity in the presence of different doses of compound 1 (0, 0.078,0.156,0.313,0.625,1.25 /jM) and 0.3 mM OMFP in 20 mM Bis-Tris, pH 6.0,150 mM NaCI, 1 mM DH, and 0.005 % Tween-20 in 20 /jL totai assay voiume in biack 384-weii microtiter plates. No time-dependent inhibition was observed as demonstrated by the linear progress curves of the HePTP phosphatase reaction, (b) Eadie-Hofstee plot of the Michaelis-Menten kinetic study with compound I.The HePTP-catalyzed hydrolysis of OMFP was assayed at room temperature in a 60 /jL 96-well format reaction system in 50 mM Bis-Tris, pH 6.0 assay buffer containing 1.7 mM DTT, 0.005 % Tween-20, and 5 % DMSO. Recombinant HePTP (5 nM) was preincubated with various fixed concentrations of inhibitor (0,0.1,0.2,0.4,0.8,1.6 /jM) for 10 min. The reaction was initiated by addition of various concentrations of substrate (0,12.5,25,50,100,200,400 pM) to the...

Again, the configuration is inverted. Nucleoside diphosphate kinase catalyzes the same transfer, but to a nucleoside diphosphate rather than to AMP, and with retention of configuration rather than with inversion (70). The mechanism of action of adenylate kinase involves a single displacement at P and that of nucleoside diphosphate kinase involves a double displacement at P via an intermediate phosphoenzyme. Although alkaline phosphatase is not classified as a phosphotransferase, it catalyzes transphosphorylation via the same phosphoenzyme that is the intermediate in the phosphatase reaction. This enzyme catalyzes reaction... 

Fig. 1. The metabolic interconversion of various forms of vitamin Bg. Reactions 1, 2 and 3 are catalysed by pyridoxal kinase, reactions 4, S and 6 by various phosphatases, reactions 7, 8 and 10 by pyridoxal-P oxidases, reactions 9 and 11 by certain aminotransferases, and reactions 12 and 13 by various pyridoxal dehydrogenases (taken from ref. 2).

When desensitization is due to some covalent modification, the particular condition (8.7) does not necessarily hold, because the direct and reverse steps in the cyclical system correspond to the direct steps of distinct reactions catalysed by different enzymes (Segel et al, 1986 Waltz Caplan, 1987). Thus, in the case of receptor phosphorylation in Dictyostelium, the constants k, 2 and k, k 2 relate, respectively, to the direct steps of the reactions catalysed by the protein kinase and by the phosphatase, reactions for which the reverse steps are being neglected. Conditions (8.6) then suffice to ensure exact adaptation for all stimuli, as indicated by fig. 8.7 and by fig. 8.8a where the changes in the activity are shown, in response to stimuli of increasing magnitude. 

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