Phosphatases linkage specificity

Enzymes that react with a specific type of ester linkage are known as general hydrolysing enzymes. Thus lipases hydrolyse a wide range of organic esters. Generally, phosphatases will break down phosphate esters into phosphoric acid and an alcohol. 

The mode of phosphorylation can also be determined enzymatically using phosphatases that are specific for either the serine/threonine or the tyrosine linkages. Also, tyrosine specific phosphorylation can be detected using phosphotyrosine specific antibodies (Kamps and Sefton 1988). 

A carbocyclic NAD(+) analogue (91) incorporating a methylenebisphospho-nate linkage in place of the natural pyrophosphate has been prepared as an inhibitor of ADP-ribosyl cyclase which is resistant to non-specific phosphatase degradation.The analogue 91 was obtained in 25% yield following a Poulter coupling of the precursor 92 with adenosine 5 -methylenebisphosphonate. 

The phosphomonoesterases that proved most useful in this work, although free of proteolytic impurities, were found to be complex in their behavior toward phosphate esters. As indicated in Table II, if tested with the aid of low molecular weight substrates, the intestinal (85) and the potato phosphatase (34) act on 0—P and N—P bonds, whereas the prostate enzyme (86) hydrolyzes only 0—P linkages. After the discovery of the specificity of two of these enzymes for low molecular weight N—P esters, it was noticed that the intestinal enzyme, although classified in the literature as alkaline phosphatase, hydrolyzes N—P bonds both at pH 5.6 and 9.0, but not at pH 7.0. Since the pH range of 5 to 6 is that of maximum stability of almost all proteins, most experiments were carried out in this pH range. Thus the use of these three enzymes, either alone or in combination with each other, proved to be quite a powerful tool. 

The observation that prostate phosphatase liberates only 40% of the a-casein phosphorus and does not act on /3-casein foreshadowed the existence of phosphodiesters and pyrophosphate bonds in these proteins. Thus the action on these materials of enzymes specific for such bonds should establish not only the nature of the linkages but also the proportions in which they occur. The results of such stepwise enzymatic analysis can be best followed with the aid of Table V. 

Enzymes modified with carbohydrates (neoglycoenzymes) can be used in cytochemistry as described above or in biochemical detection of lectins in solid-phase assays to gain greater sensitivity in analysis. For example, bacterial 3-galactosidase modified with p-aminophenyl a-D-mannopyranoside via amide linkage was useful in determination of Con A immobilized on plastic microtiter plates, and lactose-modified P-galactosidase was effective in histochemical detection of galactoside-specific lectins [63]. Other enzymes frequently used for these applications are alkaline phosphatase and horse radish peroxidase. There are a number of colorimetric, fluorometric, and chemiluminescent substrates available for these enzymes. 

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