Alkaline phosphatase reaction catalyzed

Alkaline phosphatase catalyzes the dephosphorylation of a mmber of artificial substrates ( ) including 3-glycerophosphate, phenylphosphate, p-nitrophenylphosphate, thymolphthalein phosphate, and phenolphthalein phosphate. In addition, as shown recently for bacterial and human enzymes, alkaline phosphatase simultaneously catalyzes the transphosphorylation of a suitable substance which accepts the phosphoryl radical, thereby preventing the accumulation of phosphate in the reaction mediim (25). 

An example of an optical enzyme sensor (Arnold, 1985) in a bifurcated optical fiber is shown in Fig. 9.32. The bifurcated fiber delivers and collects light to and from the site of the enzymatic reaction. The enzyme, alkaline phosphatase (AP), catalyzes hydrolysis of p-nitrophenyl phosphate to p-nitrophenoxide ion which is being detected (A = 404 nm). 

Fig. 6 The catalytic mechanism of the alkaline phosphatase reaction. The initial alkaline phosphatase (E)-catalyzed 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 step of the reaction, phosphate is released through hydrolysis of the covalent intermediate (E-Pi) and the non-covalent complex (E Pi) of the inorganic phosphate in the active site. In the presence of nitrogen-containing alcohol molecules (AOH), such as the buffer diethanolamine (DEA), phosphate is also released via a transphosphorylation reaction...

The first absorbance-based fiber-optic biosensor was that for p-nitrophenylphosphate (8). This biosensor uses the enzyme alkaline phosphatase which catalyzes the following reaction ... 

Numerous investigations of phosphate esters hydrolysis continue to be reported. The nonenzymatic and alkaline phosphatase (AP) catalyzed rates of hydrolysis for a series of substituted methyl phenyl phosphate diesters have been measured. The result obtained suggest that reactions catalyzed by (AP) proceed through a synchronous transition state that is indistinguishable from that in solution, even though the AP active site is optimized for the loose transition state of the phosphate... 

Craig D B, Arriaga E A, Wong J C Y, Lu H and Dovichi N J 1996 Studies on single alkaline phosphatase molecules reaction rate and activation energy of a reaction catalyzed by a single molecule and the effect of thermal denaturation—the death of an enzyme J. Am. Chem. See. 118 5245-53... 

Enzymes are powerful catalysts. Enzyme-catalyzed reactions are typically 10 to times faster than their uncatalyzed counterparts (Table 16.1). (There is even a report of a rate acceleration of >10 for the alkaline phosphatase-catalyzed hydrolysis of methylphosphate )... 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

Product Acceptors. Many enzyme assays use acceptors, as for instance 2-ethylaminoethanol and other aminated alcohols iihich act as acceptors for the phosphoryl product of the reaction catalyzed by alkaline phosphatase (25) (Fig. 4). Hydroxylamine can act as an acceptor for the hydroxyacetone produced by eno-lase and semicarbazide can act as an acceptor for the pyruvate produced by LD. It is necessary to optimize the concentration of such an acceptor before using it routinely as often what may be a theoretically desirable acceptor is in practice superfluous. 

One of the most important metals with regard to its role in enzyme chemistry is zinc. There are several significant enzymes that contain the metal, among which are carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, aldolase, and carbonic anhydrase. Although most of these enzymes are involved in catalyzing biochemical reactions, carbonic anhydrase is involved in a process that is inorganic in nature. That reaction can be shown as... 

Recently, two major enzyme-catalyzed chemiluminescent reactions have become popular. These use either luminol as a substrate of peroxidase or 3-(2 -spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) as a substrate of alkaline phosphatase (ALP). 

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. 

These enzymes, which mainly catalyze hydrolytic reactions, have the zinc ions at their active sites. However, Zn ions also appear necessary in some cases for stabilization of the protein structure, e.g. in Cu/Zn SOD, insulin, liver alcohol dehydrogenase and alkaline phosphatases. 

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,... 

Enzymes are biocatalysts constructed of a folded chain of amino acids. They may be used under mild conditions for specific and selective reactions. While many enzymes have been found to be catalytically active in both aqueous and organic solutions, it was not until quite recently that enzymes were used to catalyze reactions in carbon dioxide when Randolph et al. (1985) performed the enzyme-catalyzed hydrolysis of disodium p-nitrophenol using alkaline phosphatase and Hammond et al. (1985) used polyphenol oxidase to catalyze the oxidation of p-cresol and p-chlorophenol. Since that time, more than 80 papers have been published concerning reactions in this medium. Enzymes can be 10-15 times more active in carbon dioxide than in organic solvents (Mori and Okahata, 1998). Reactions include hydrolysis, esterification, transesterification, and oxidation. Reactor configurations for these reactions were batch, semibatch, and continuous. 

Sucrose phosphate synthetase catalyzes the reaction of UDP-glucose with fructose-6-P to form sucrose-6-P and UDP. This step is the penultimate step in the synthesis of sucrose in leaves. The chromatographic method can be applied to many UDP-glucose-requiring enzymes. The method eliminates the need for treatment of reaction mixtures with alkaline phosphatase. 

Alkaline phosphatases are typically dimeric zinc metalloenzymes ranging in size from 80 to 145 kDa. They catalyze a nonspecific phosphomonoesterase reaction of the following type ... 

Other enzymes are also useful indices of liver pathology. Serum alkaline phosphatase is often a useful indicator of liver and bone disease. The alkaline phosphatases are a diverse group of enzymes that catalyze reactions in which a phosphate is removed from a phosphate ester, especially at an alkaline pH. Physicians don t care about this. They do care that serum alkaline phosphatase levels often rise with bone breakdown (as in tumor infiltration) and in liver disease, especially where tliere is obstruction of the bile duct. Acid phosphatase is particularly rich in the prostate. A rise in its serum levels provides a test as to the presence of prostate carcinoma. This test has largely been replaced by assay for Prostate Specific Antigen (PSA), a serine protease that is elevated in prostatic carcinoma. 

Detection limits in EIA are ultimately determined by how low one can measure the label s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, /8-galactosi-dase, peroxidase, - and glucose oxidase. This is due to the high mo-... 

The direct detection of electrochemical labels entails problems with sensitivity. For this reason the majority of electrochemical immimoassay development has focused on the measurement of enzyme labels by detection of eiectroactive products arising from enzyme catalyzed reactions. A wide variety of enzyme labels have been used for electrochemical immunoassays. These include glucose oxidase, glucose-6-phosphate dehydrogenase and alkaline phosphatase. ... 

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