Acid phosphatase kinetics

Ascencio, J. (1997) Root-secreted acid phosphatase kinetics as a physiological marker for phosphorus deficiency, youma/ of Plant Nutrition 20, 9-26. 

AGIRE computer program for, 249, 79-81, 225-226 comparison to analysis based on rates, 249, 61-63 complex reactions, 249, 75-78 experimental design, 249, 84-85 inhibitor effects, 249, 71-75 potato acid phosphatase product inhibition, 249, 73-74 preliminary fitting, 249, 82-84 prephenate dehydratase product inhibition, 249, 72-73 product inhibition effects, 249, 72-73 prostate acid phosphatase phenyl phosphate hydrolysis, 249, 70 reactions with two substrates, 249, 75-77 reversible reactions, 249, 77-78 with simple Michaelian enzyme, 249, 63-71 [fitting equations, 249, 63] with slow-binding inhibitors, 249, 88 with unstable enzymes, for kinetic characterization, 249, 85-89. 

The iron(II)-iron(III) form of purple acid phosphatase (from porcine uteri) was kinetically studied by Aquino et al. (28). From the hydrolysis of a-naphthyl phosphate (with the maximum rate at pH 4.9) and phosphate binding studies, a mechanism was proposed as shown in Scheme 6. At lower pH (ca. 3), iron(III)-bound water is displaced for bridging phosphate dianion, but little or no hydrolysis occurs. At higher pH, the iron(III)-bound OH substitutes into the phosphorus coordination sphere with displacement of naphthoxide anion (i.e., phosphate hydrolysis). The competing affinity of a phosphomonoester anion and hydroxide to iron(III) in purple acid phosphatase reminds us of a similar competing anion affinity to zinc(II) ion in carbonic anhydrase (12a, 12b). 

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. 

Two major difficulties must be considered in any assay for acid phosphatase. The enzyme is subject to surface inactivation (23, 24). Accordingly, reproducible initial hydrolytic rates are not always obtained, and the kinetic behavior should be checked in any new assay developed. Discrepancies between the amount of inorganic phosphate produced and phenol liberated from phenolic phosphates may be substantial if extensive phosphotransferase activity occurs because of phosphoryl acceptor action on the part of hydroxylic buffers or other constituents of the incubation mixture (25, 26). Fluorogenic assays have been developed with very high sensitivity (27). Reference will be made to particular assays in the discussion of the specific enzymes. 

Effect of Substrate and Buffer on Kinetic Constants of Acid Phosphatase ... 

Solutions of acid phosphatase are particularly sensitive to surface inactivation. Figure 3 (88) shows the inactivation rate of the enzyme in the presence and absence of surface-active detergents. The inactivation process is temperature sensitive and the protection by detergent is total. Most of the enzyme inactivation proceeds with first-order kinetics. A variety of agents—gelatin, bovine serum albumin, egg albumin, and Tween-80—protect the enzyme against inactivation. 

Reiner and his colleagues (40) demonstrated that fluoride inhibition of prostatic acid phosphatase showed interesting and unexpectedly complex kinetics. The unusual nature of the inhibition can readily be appreciated from Fig. 4 (40). As the fluoride concentration is increased over a 1000-fold range, the extent of inhibition rises and then subsequently falls with a further increase of inhibitor. At lower fluoride concentration, the inhibition is clearly competitive. Two possibilities were explored for an explanation of these unusual concentration effects of inhibition. There could be two forms of fluoride in the reaction mixtures the inhibitory form and the second which predominates at higher... 

Vescia and Chance Hi) demonstrated that fluoride and tartrate inhibition vide infra) of acid phosphatase showed completely different kinetics when the hydrolysis of phenyl phosphate was compared with transphosphorylation from this substrate to glucose. Figures 5 and 6 (41) show that fluoride inhibition is competitive when the data are plotted according to Lineweaver and Burk. However, the inhibition is noncompetitive with respect to transphosphorylation of the same substrate to glucose. The authors suggested that there are two distinct sites... 

Prostatic acid phosphatase is partially and reversibly inactivated by calcium ion (45). Anions such as chloride, bromide, and thiocyanate inhibit prostatic acid phosphatase competitively with regard to substrate as well as noncompetitively. A kinetic analysis by London et al. (46) indicates that the noncompetitive inhibition was related to changes in charge on the protein molecule. A variety of nonspecific anions accelerate thermal denaturation of the enzyme. The enzyme is quite sensitive to a number of electrolyte changes, but it is not clear whether these factors are involved in biological control. 

Scott (80) purified red cell acid phosphatase of homozygous types A and B by using ammonium sulfate and DEAE-cellulose chromatography. The relative activity of these isozyme preparations was the same when tested with a number of substrates. Type B enzyme showed small kinetic... 

Various kinetic studies of the bovine spleen acid phosphatase have been reported (Vincent etal., 1991). The results are consistent with a picture in which the oxyanions bind in a non-competitive fashion by bridging the two iron atoms in the PAP s dinuclear centre, with the smaller anions also able to bind in a competitive manner at a second site (Fig. 5-21). 

In general there are three phosphatase families alkaline, acid, and protein phosphatases. Alkaline phosphatases are typically dimers that contain three metal ions per subunit and have a pH optimum pH above 8. Acid phosphatases exhibit an optimum pH<7 and are usually divided into three classes low molecular weight acid phosphatases (<20 kDa), high molecular weight acid phosphatases (50-60 kDa), and purple acid phosphatases (which contain an Fe-Fe or Fe-Zn center at the active site). Phosphatases specific for I-l-P appear to be most similar (in kinetic characteristics but not in mechanism) to the alkaline phosphatases, but their structures define a superfamily that also includes inositol polyphosphate 1-phosphatase, fructose 1, 6-bisphosphatase, and Hal2. The members of this superfamily share a common structural core of 5 a-helices and 11 (3-strands. Many are Li+-sensitive (York et al., 1995), and more recent structures of archaeal IMPase proteins suggest the Li+ -sensitivity is related to the disposition of a flexible loop near the active site (Stieglitz et al., 2002). 

Using an approximately 300-fold purified preparation of prostatic acid phosphatases, obtained essentially according to the procedure of London and Hudson (LIO), Tsuboi and Hudson (T3) undertook several types of kinetic studies. These investigators observed that the purified preparation of the prostatic acid phosphatase was highly unstable in... 

N3. Nigam, V. N., Davidson, H. M., and Fishman, W. H., Kinetics of hydrol5rsis of the orthophosphate monoesteis of phenol, p-nitrophenol and glycerol by human prostatic acid phosphatase. J. Biol. Chem. 234, 1550-1554 (1959). 

Scott, E. M., Kinetic comparisons of genetically different acid phosphatases of human erythrocytes. J. Biol. Chem. 241, 3049-3052 (1965). 

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). 

Fig. 20 The reaction mechanism of acid phosphatases implicated by structural, kinetic, and...

Nakanishi M, Yoh K, Miura T, Ohasi T, Rai SK, Uchida K. Development of a kinetic assay for band 5b tartrate-resistant acid phosphatase activity in serum. Clin Chem 2000 46 469-73. 

Luchter-Wasylewska E (2001) Cooperative kinetics of human prostatic acid phosphatase. Biochim Biophys Acta 1548 257-264... 

Valcour AA, Bowers GN Jr, McComb RB (1989) Evaluation of a kinetic method for prostatic acid phosphatase with use of self-indicating substrate, 2,6-dichloro-4-nitrophenyl phosphate. Clin Chem 35 939-945... 

After the specimen has been applied to the slide, a distributor arm moves the slide to the proper incubator CM for the colorimetric and two-point rate enzyme tests (acid phosphatase, amylase, and lipase), PM for the potentiometric chemistries, and RT for the rate or kinetic incubator for the multiple-point rate enzyme chemistries. Temperature control within either the CM or RT incubator is maintained at 3 7 0.1 ° C by contact of the slide with the rotating thermal mass of the incubator. The products forming in the slides in either the CM or RT incubator are monitored at what are termed read stations by separate reflectance densitometers or reflectometers. There are, however, differences on how such measurements are made. For the enzyme slides in the CM incubator, at selected... 

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