Phosphatase phosphomonoesterase

Increased soil phosphatase activity, net plant primary production, total aboveground P Increased phosphomonoesterase activities, increased soil and shoot P... 

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. 

Acid phosphatase (acid phosphomonoesterase, EC 3.1.3.2) also catalyzes the hydrolysis of phosphoric acid monoesters but with an acidic pH optimum. It has broad specificity and catalyzes transphosphorylations. Acid phosphatases are a quite heterogeneous group with monomeric, dimeric, larger glycoprotein, and membrane-bound forms. Acid phosphatase activity is present in the heart, liver, bone, prostate, and seminal fluid. Prostate carcinomas produce large quantities of acid phosphatase, and the enzyme is, therefore, used as a biomarker [141]. 

This enzyme [EC 3.1.3.2], also referred to as acid phos-phomonoesterase, phosphomonoesterase, and glycero-phosphatase, catalyzes the hydrolysis of an orthophos-phoric monoester to generate an alcohol and... 

Another phosphomonoesterase family, the purple acid phosphatases, have been attracting interest, since they contain a mixed-valence binu-clear iron(II/III) center (26). Although the exact roles of iron(II) and iron(III) have not been clarified yet, it has recently been reported that the direct nucleophilic attack of Fe111—OH- at the phosphate P atom is the most likely mechanism (27). 

Milk contains several phosphatases, the principal ones being alkaline and acid phosphomonoesterases, which are of technological significance, and ribonuclease, which has no known function or significance in milk. The alkaline and acid phosphomonoesterases have been studied extensively (see Andrews (1993) for references). 

Alkaline phosphomonoesterase (EC 3.1.3.1). The existence of a phosphatase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacterium tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed, and a test procedure based on alkaline phosphatase inactivation was developed for routine quality control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenyl-phosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively ... 

Acid phosphomonoesterase (EC 3.1.3.2). Milk contains an acid phosphatase which has a pH optimum at 4.0 and is very heat stable (LTLT pasteurization causes only 10-20% inactivation and 30 min at 88°C is required for full inactivation). Denaturation of acid phosphatase under UHT conditions follows first-order kinetics. When heated in milk at pH 6.7, the enzyme retains significant activity following HTST pasteurization but does not survive in-bottle sterilization or UHT treatment. The enzyme is not activated by Mg2+ (as is alkaline phosphatase), but it is slightly activated by Mn2+ and is very effectively inhibited by fluoride. The level of acid phosphatase activity in milk is only about 2% that of alkaline phosphatase activity reaches a sharp maximum 5-6 days post-partum, then decreases and remains at a low level to the end of lactation. 

Horiuchi et al. (2), and Torriani (S) that orthophosphate repressed the formation of a nonspecific phosphomonoesterase in E. coli that research on this enzyme began. This work (2, 3) showed a maximum rate of synthesis of the enzyme occurred only when the phosphate concentration became low enough to limit cell growth. With sufficient phosphate, the amount of active enzyme is negligible. Under conditions of limiting phosphate, alkaline phosphatase accounts for about 6% of the total protein synthesized by the cell (4). 

Alkaline and acid phosphatase are organ-specific enzymes that are assayed in the diagnosis of many diseases. These activities are phosphomonoesterases that dephosphorylate a number of compounds, including nucleoside monophosphates, to their respective nucleosides and free phosphates. However, such dephosphorylations have traditionally been assayed with 4-nitrophenyl... 

Figure 9.93 HPLC chromatograms of phosphomonoesterase hydrolysis of A(S)MP. (i4) Chromatogram obtained from calf intestinal mucosa alkaline phosphatase hydrolysis of A(S)MP. In a reaction volume of 100 /xL containing 100 mM Tris-HCl (pH 8.1), 300 pM A(S)MP, and 20 mM MgCl2, the reaction was initiated by addition of 2 of enzyme and incubated at 30°C for 6 hours. A 20 /xL sample was then injected onto the HPLC column and analyzed. (B) Chromatogram obtained from snake venom S -nucleotidase incubated with A(S)MP. In a reaction volume of 100 /xL containing 100 mM Tris-Cl (pH 8.1), 300 jxM A(S)MP, and 20 mM MgCl2, the reaction was initiated by addition of 6 yxg of enzyme and the reaction mixture incubated at 30°C for 60 minutes, and a 20 yxL sample was injected onto the HPLC column and analyzed. (From Rossomando et al., 1983.)...

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 ... 

An approximate idea of the distribution of acid phosphatase activity in human tissues, regardless of the nature of the acid phosphatase, may be obtained from the studies of Reis (R2) on 5 -nucleotidase and other phosphomonoesterases. He prepared aqeuous homogenates of postmortem tissue in the proportion of 20 parts of water to one of tissue, allowed these to autolyze for 2 days at room temperature, centrifuged the material, and employed the supernatant fluid. The assay mixture consisted of 0.4 ml of a suitable buffer, 0.1 ml of 0.005 Af phenyl phosphate as substrate, and 0.1 ml of tissue extract. The enzyme activity was expressed as micrograms of phosphorus hydrolyzed per hour per milligram of wet... 

T3. Tsuboi, K. K., and Hudson, P. B., Acid phosphatase. III. Specific kinetic properties of highly purihed human prostatic phosphomonoesterase. Arch. Biochem. Biophys. 55, 191-205 (1955). 

Gulland and Jackson confirmed that phosphomonoesterase liberates 7%, or less, of the phosphoric acid from ribosenucleic acid. They therefore concurred with Takahashi that this kind of ribosenucleic acid contains no phosphomonoester group and that each phosphorus atom is present as a disubstituted phosphoryl group, but they pointed out that this is not necessarily a di-ester group. The liberation of 7%, or less, of the total phosphorus is in accord with the idea of a polymerized tetranucleotide 25% of the phosphorus in the simple tetranucleotide (VII) should be hydrolyzable by phosphatase. 

Dephosphorylation may be carried out using bacterial alkaline phosphomonoesterase (Heppell et al. 1962). Typical conditions are 20 fig desalted oligonucleotide mixture in 2 ml 1 M ammonium bicarbonate with 10 fig enzyme incubated for 1 hr at 37°C (De Wachter and Piers 1967). This removes 3 -terminal phosphates. Brownlee and Sanger (1967) carried out combined T1 ribonuclease and bacterial alkaline phosphatase digestion of the low molecular weight rRNA using enzyme/substrate ratios of 1/20 and 1/5 respectively in 0.01 M Tris pH 8.0 at 37°C for 1 hr. This produces fragments like XpXp...XpG where X represents any nucleoside except G. 

Considerations of this kind led the author to undertake an investigation using enzymes to reveal the chemical nature of phosphorus bonds that may occur in phosphoproteins. This interest came through the accidental observation that a variety of phosphomonoesterases of mammalian origin and from plants will dephosphorylate ovalbumin, a protein with a low phosphorus content. Of course, a prerequisite in the selection of the enzymes for such work is that the dephosphorylation process should not be accompanied by any other enzymatic reactions that might result from the presence of small amounts of impurities in even highly purified phosphatase preparation. in particular, an extensive proteolysis has to be excluded. 

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. 

In a recent communication Sundararajan and Sarma report that a phosphoprotein phosphatase from rat spleen dephosphorylates a-, /3-, and unfractionated casein (90). Since these authors state that their enzyme differs in its action from that of a phosphomonoesterase, their results are in accord with the occurrence of a variety of phosphorus bonds in proteins. In this connection it should be noted that intestinal phosphatase used in our work at pH 9.0 also liberates all of the a-casein phosphorus (72). As discussed earlier, although this enzyme at pH 6.0 hydrolyzes —N—P—... 

Utilization of phosphate monoesters by microalgae and bacteria is effected by phosphomonoesterases (phosphatases) of broad specificity present at the cell surface. Hydrolytic release of PO4- from sugar phosphates, nucleotide phosphates, phospholipids, and phenyl phosphates, to name a few, enables a wide variety of phosphorus containing compounds to be utilized as phosphorus sources for growth of microbes. Ultrastructural observations and results from biochemical experiments indicate that extracellular phosphatases cleave the phosphate moiety from dissolved organic phosphorus compounds, which is then internalized, leaving the carbon skeleton outside the cell (Kuenzler and Perras, 1965 Doonan and Jensen, 1977). 

Alkaline phosphatases (AP EC 3.1.3.1) are dimeric, zinc-containing, non-specific phosphomonoesterases which are found in... 

In rabbit iris sphincter smooth muscle microsomal fraction, there are phosphomonoesterases that degrade IP3 to IP2, IP2 to IP, and IP to free myo-inositol and Pj (Abdel-Latif, 1986). The IP3 phosphatase has been shown to specifically remove the 5-phosphate from IP3 and from cyclic IP3 to produce IP2 and cyclic IP2, respectively. The polyphosphoinositide phosphatase has also been reported to dephosphorylate inositol tetrakisphophate (IP4) to IP3. These enzyme activities are both cytosolic and membranous, dependent on Mg2+, and not inhibited by Li+. The IP3 5-phospha-tase was studied in the microsomal fraction of bovine iris sphincter muscle (Wang et al., 1994). It hydrolyzed IP3 to I(1,4)P2 with an apparent of 28 (xM Mg2+ was required for its activity, Ca + (> 0.5 (xM) was inhibitory, and Li+ or phosphorylation of the microsomal fraction with cAMP-dependent protein kinase or protein kinase C (PKC) had no effect on the activity of the enzyme. 

From these data it appears that through the action of specific inositol phosphatases both IP3 and IP4 are sequentially dephosphorylated to free inositol (cf. Fig. 2). The dephosphorylation of IP3 requires Mg2+ and physiological concentrations of Ca +. The inositol phosphate phosphomonoesterase is inhibited by Li+, but the IP3 5 -phosphomonoesterase is not inhibited (Carsten and Miller, 1990). Interestingly, soluble and particulate extracts from porcine skeletal muscle also metabolize IP3 and IP4 to inositol in a stepwise fashion (Foster et al., 1994). Apparently, smooth and skeletal muscles have the same set of inositol polyphosphate phosphatases, although their functional role in skeletal muscle is not known. 

Enzymatic reactions can be used as a means of both characterizing and releasing classes of organic phosphorus present in waters, soils and sediments. For example, phosphate monoesters can be quantified by the use of a phosphomonoesterase such as alkaline phosphatase. Alternately, the high specificity of some enzymes for particular substrates can be used as the basis for determination of specific organic phosphorus compounds, or as part of a post-separation quantification step. 

Many living cells contain at least two types of phosphomonoesterase enzymes (EC 3.1.3), the acid and the alkaline phosphatases. 

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