Phosphate acceptors

The phosphorylation and nucleotide binding domains. The phosphorylation domain contains the phosphate acceptor Asp351, that is phos-phorylated by ATP during Ca " " transport [45,80,87,94,95]. Site-specific... 

Although the sequence identity averaged over the whole length of the molecule is generally low among different P-type ion transport ATPases, the conserved sequences around the phosphate acceptor aspartyl group and in the ATP binding domain are well preserved [30,32,46]. Structure predictions based on the hydropathy plots... 

A similar mechanism was postulated for the Ca " -dependent inactivation of Ca -ATPase by ATP-imidazolidate [380] that results in intramolecular crosslinking with the formation of a new protein band of 125 kDa. In both cases the reactive carboxyl group was suggested to be the phosphate acceptor Asp351. 

AP isoenzymes can cleave associated phosphomonoester groups from a wide variety of substrates. The exact biological function of these enzymes is not well understood. They can behave in vivo in their classic phosphohydrolase role at alkaline pH, but at neutral pH AP isoenzymes can act as phosphotransferases. In this sense, suitable phosphate acceptor molecules can be utilized in solution to increase the reaction rates of AP on selected substrates. Typical phosphate acceptor additives include diethanolamine, Tris, and 2-amino-2-methyl-lpropanol. The presence of these additives in substrate buffers can dramatically increase the sensitivity of AP ELISA determinations, even when the substrate reaction is done in alkaline conditions. 

An interesting approach towards the formation of glycosyl 1-phosphates by dehydrative glycosylation of nucleophilic dialkyl phosphate acceptors with... 

Phosphates of pharmaceutical interest are often monoesters (Sect. 9.3), and the enzymes that are able to hydrolyze them include alkaline and acid phosphatases. Alkaline phosphatase (alkaline phosphomonoesterase, EC 3.1.3.1) is a nonspecific esterase of phosphoric monoesters with an optimal pH for catalysis of ca. 8 [140], In the presence of a phosphate acceptor such as 2-aminoethanol, the enzyme also catalyzes a transphosphorylation reaction involving transfer of the phosphoryl group to the alcohol. Alkaline phosphatase is bound extracellularly to membranes and is widely distributed, in particular in the pancreas, liver, bile, placenta, and osteoplasts. Its specific functions in mammals remain poorly understood, but it seems to play an important role in modulation by osteoplasts of bone mineralization. 

This enzyme is notable in that it is not specific for the base (purines or pyrimidines) or the sugar (ribose or deoxyribose). This nonspecificity applies to both phosphate acceptor (A) and donor (D), although the donor (NTPD) is almost invariably ATP, because it is present in higher concentration than other nucleoside triphosphates under aerobic conditions. 

When guanosine 2, 3 -cyclic phosphate is incubated with about 3-fold nucleoside at a low temperature in the presence of RNase N, guanylyl-(3, 5 )-nucleoside can be obtained as a synthetic product. For example, using uridine as a phosphate acceptor, GpU was obtained with a high yield of 27% calculated upon guanosine 2, 3 -cyclic phosphate added. This... 

Since the equilibrium lies well to the right it is customary to say that alkaline phosphatase hydrolyzes phosphate esters, but some related compounds are also hydrolyzed (Table VI) 3, 4, 28, SO, 94-100). The enzyme also catalyzes transphosphorylation reactions in which a different alcohol substitutes for H20 as a phosphate acceptor. Compounds that are hydrolyzed have the general structure,... 

Scheme I shows the hydrolysis of a phosphate ester in the presence of tris, which can serve as a phosphate acceptor so that O-phosphoryl-tris is a product as well as P(. It has been shown that in the presence of alcohols such as tris and ethanolamine the rate of substrate utilization is increased, that formation of alcohol exceeds that of phosphate, and that the difference is due to the formation of the O-phosphorylamino alcohol (122, 128). The question was Does the reaction with water and with tris emanate from the Michaelis complex or from a phosphoryl enzyme intermediate (E-P) If the reactions with tris and water stem from a phosphoryl enzyme, the ratio of products tris-phosphate and Pi would be independent of the leaving group RO, but if the reactions stem from the reversible complex containing the leaving group, the ratio of products would depend upon the structure of R. It was found that the ratio of free alcohol to phosphate was 2.39 0.02 for nine different substrates, including esters such as p-cresyl phosphate / -naphthyl phosphate, and phosphoenol pyruvate. This experiment established the occurrence of a phosphoryl enzyme intermediate.

Many other methods have been employed to study CTC in biological systems, such as calorimetry, mixed fusion analysis, solubility and partition methods, ultrasonic methods, spectropolarimetry, reflective infrared spectroscopy, Raman spectroscopy, flash photolysis spectroscopy, nuclear quadrupole resonance spectroscopy, and magnetic susceptibility methods, to name several of a very long list. X-ray photoelectron spectroscopy (XPS) has also been used to elucidate some EDA interactions in electrically active macromolecules. XPS is useful for detecting the redistribution of charges in complexes of such compounds, (e.g., in the presence of phosphate acceptors, the nature of the semiconductive environment of S, O, and N bridges in macromolecules is affected profoundly [111]. 

Another enzyme that uses the energy of the PPj is the pyruvate, phosphate dikinase. This enzyme converts P-enolpyruvate to pyruvate, but unlike pyruvate kinase, which uses ADP as the phosphate acceptor yielding ATP and pyruvate. This enzyme uses AMP and in combination with PPj yields ATP, Pj and pyruvate ... 

One of the most widespread examples of phenylethanoid glycosides is verbascoside, also called acteoside, which has been obtained form sources belonging to different plant families. This compound, isolated from Lantana camara (Verbenaceae), was characterised as an inhibitor of rat brain PKC, with an IC50 value of 25 iM. This effect was abolished by adding ATP, which indicated a competitive interaction with the nucleotide. The inhibition was non-competitive with respect to the phosphate acceptor, histones type IIIS in this case, but other kinases, such as PTK from a lymphoma cell line or PKA, were not inhibited. In order to translate the biochemical effect to a related cellular event, the ability of verbascoside to reduce the proliferation of the lymphocytic mouse leukaemia L-1210 cells was examined. This compound showed an IC50 value of 13 pM [32]. 

The benzophenanthridine alkaloid chelerythrine (37) isolated from Zanthoxylum simulans (Rutaceae) is a potent, selective antagonist of PKC from rat brain (IC50 = 0.66 pM). Inhibition was competitive with respect to the phosphate acceptor (histone Ills) and non-competitive with respect... 

ALP catalyzes the hydrolysis of 4-NPP, forming phosphate and free 4-nitrophenol (4-NP, PNP), which in dilute acid solutions is colorless. Lfnder alkaline conditions, 4-NP is converted to the 4-nitrophenoxide ion, which has a very intense yellow color. The rate of formation of 4-NP by the action of the enzyme on 4-NPP at 37 °C is then monitored at 405 nm with a recording spectrophotometer. The (provisional) IFCC-recommended method uses 4-NPP as the substrate and AMP as the phosphate-acceptor buffer. It... 

Continuous-monitoring methods for assay of TR-ACP activity are based on the principle introduced by Hillmann in which a-naphthoi released from its phosphate ester forms a colored product with the stabilized diazonium salt of 2-amino-5-chlorotoluene-1,5-naphthalene disulfonate (Fast Red TR). The introduction of alcohols, such as 1,5-pen-tanediol, accelerates the reaction and increases sensitivity by acting as phosphate acceptors in transfer reactions. The addition of sodium tartrate inhibits the sensitive isoenzymes (i.e., prostatic and lysosomal ACPs) if they are present in the sample. 

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