Acid phosphatase sweet potato enzyme

Presently, the vast majority of information on the Mn site in these acid phosphatases comes from the enzyme from sweet potato tubers. This 110-kDa enzyme is identical to the previously reported sweet potato enzyme likewise, a 55-kDa subunit was found (78). However, the enzyme possesses only one Mn per enzyme molecule. At 293 and 77 K, no EPR signal could be detected for the native enzyme. Inactivation of the enzyme by heat treatment or the addition of acid results in the appearance of a six-line EPR pattern due to aquated Mn(II). As in the case of Mn SODs, this was taken as evidence for Mn(III) in the native... 

Purple acid phosphatase (PAP) or tartrate-resistant phosphatase is not thought to be a protein phosphatase but it has a very similar dimetallic active site structure to that found in protein phosphatases. PAPs have been identified in bacteria, plants, mammals, and fungi. The molecular weights (animal 35 kDa, plant 55 kDa) are different and they exhibit low sequence homology between kingdoms but the residues involved in coordination of the metal ions are invariant. " There has been considerable debate as to the identity of the metal ions in PAPs in vivo. Sweet potato, Ipomoea batatas, has been shown to possess two different PAP enzymes and the active site of one of them has been shown to contain one Fe and one Zn " " ion. Another report has established that the active site of a PAP from sweet potato contains one Fe " and one Mn +. The well-characterized red kidney bean enzyme and the soybean enzyme contain Fe " and Zn. Claims that PAP from sweet potato has 2Fe ions or 2Mn ions have been discussed elsewhere. One explanation is that these are different forms of the enzyme, another is that because the metal ions are labile and are rapidly incorporated into the active site, the enzyme contains a mixture of metal ions in vivo and the form isolated depends on the conditions of isolation. 

Some acid phosphatases from animals and plants are violet in color and contain iron (Chapter 16) and an Mn3+-containing acid phosphatase has been isolated from sweet potatoes.720 These enzymes have dimetal centers, often containing one Zn2+ and one Fe3+ with bridging carboxylate and hydroxide ions between the metals. Imidazole, tyrosinate, and carboxylate side chains hold the metals as in Fig. 16-20. A water molecule bound to the Fe3+ is thought to dissociate with a low pKa of 4.8 to give an Fe3+ OH complex. The hydroxyl ion can then attack the phospho groups, one... 

Purple, iron-containing acid phosphatases have been purified from animal sources and from some plant sources.350 However, the purple acid phosphatase from the sweet potato contains manganese, the purple colour arising from an intense absorption band at about 515 nm. There is some doubt over the stoichiometry, in that the dimeric enzyme may contain one351 or two352 Mn2+, apparently depending on the variety of sweet potato. The iron acid phosphatases contain two Fe atoms. 

The isolation of the first manganese-containing acid phosphatase was reported in 1971 from the juice of the sweet potato (Kokei No. 14) (67). The enzyme was unique in that it was distinctly purple, the color resulting from a broad absorption band with a maximum at 555 nm. The enzyme was determined to be 110 kDa, composed of two 55-kDa subunits. The purple enzyme was capable of hydrolyzing a variety of biologically relevant phosphates as well as inorganic pyrophosphate [Eq. (2)]. Emission spectroscopy revealed the presence of Mn (68). 

Similar Mn-containing enzymes were subsequently isolated from other plant sources spinach leaves (71), rice plant cultured cells (72), soybeans (73-75), and the tubers of the sweet potato Kintoki (76-81) (Table III). Sweet potatoes have recently been reported to possess two different acid phosphatases which were immunologically distinct but which have similar molecular weights and metal content (106). Interestingly, sulfhydryl reagents have been shown to inactivate the soybean enzyme (75). 

The amino acid compositions of the beta-amylase from sweet potato, soya bean, wheat, and malted sorghum have been determined, and are shown in Table XIII. These results may suggest that the beta-amylase from these different sources differs in structure. This is, perhaps, not surprising, but it should be noted that the sorghum amylase contained 9% of pentose, and the important, sulfiir-containing amino acid cysteine was not reported and the soya-bean enzyme still contained traces of aipba-amylase and phosphatase. The sequence of amino acids, or the three-dimensional structure of any... 

The purple acid phosphatases (PAP) catalyze the hydrolysis of phosphate esters under acidic pH conditions (pH optimum 5) (9, 10). They differ from other acid phosphatases in having a distinct purple color due to the presence of iron or manganese and in being uninhibited by tartrate. Diiron units have been found in the active sites of the enzymes from mammalian spleen (171-173) and uterus (173, 174), while a heterodinu-clear FeZn unit has been characterized for the enzyme from red kidney bean (175). Either the Fe2 or the FeZn unit is catalytically competent in these enzymes, since the enzymes from porcine uterus and bovine spleen can be converted into active FeZn forms and the kidney bean enzyme can be transformed into an active Fe2 form (176). There are also enzymes from other plant sources (particularly sweet potato) that have been reported to have either a mononuclear Mn(III) or Fe(III) active site (177), but these are beyond the scope of the review. This section will focus on the enzymes from porcine uterus (also called uteroferrin), bovine spleen, and red kidney bean. 

Purple Acid Phosphatases. Purple acid phosphatases (PAPs) utilize a dinuclear metal center to catalyze the hydrolysis of phosphate monoesters. The characteristic purple color of these enzymes arises from a charge transfer absorption at about 560 nm, between a tyrosinate ligand and the conserved Fe + found in all PAPs. The second metal ion varies with the source of the enzyme and is always divalent. Mammalian PAPs are monomeric and have Fe -Fe " centers, whereas most plant PAPs are dimeric with Fe " -Zn + centers. A PAP isolated from sweet potato contains an Fe +-Mn + center, the first of its kind in any enzyme (26,27). This novel PAP also differs from others by its greater catalytic efficiency toward both activated and unactivated substrates (27), as well as in its strict requirement for manganese in the divalent site (26). 

The X-ray structures have been reported for the PAP from kidney bean (29), rat bone (30), pig (31), and sweet potato (32). Despite little sequence similarity, the enzymes share very similar catalytic sites with seven invariant amino acid ligands to the metal center (Fig. 7). The structnre of the kidney bean enzyme shows the two metal ions at a distance of 3.1 A, with a monodentate bridging Asp 164. The PAPs are related to the much larger group of Ser/Thr phosphatases (described in the next section) that share a sequence motif containing most of the residues coordinating the metal center at the active site. 

In addition to its archetypical members, uteroferrin and bovine spleen add phosphatase, the class of purple add phosphatases includes proteins isolated from rat bone and. spleen spleens of patients with Gaucher s disease or leukemic reticuloen-dotheliosis equine uterine flushings bovine cortical bone giant ceU tumors human placenta and microorganisms . The plant enzymes include an Fe-Zn phosphatase from red kidney beans and an Fe-Fe or Mn(in) protein from sweet potato tubers . Although less well-defined and more heterogeneous than their mammalian counterparts, the color and iron content of the plant enzymes warrant their designation as purple acid phosphatases. 

Although reduction and activation are synonymous for the vast majority of the purple acid phosphatases, several exceptions exist. The Fe-Zn forms of uteroferrin and bovine spleen phosphatase do not require prior reduction to exhibit enzymatic activity . The Fe-Cu and Fe-Hg derivatives of uteroferrin also do not require activation and in fact, the Fe-Cu preparation is inactivated by reducing agents The recently described high molecular weight pink form of uteroferrin has enzymic properties identical to those of purple uteroferrin treated with 2-mercaptoethanoP. Finally, the sweet potato acid phosphatase, which may exist as an 02 dimer with separate mononuclear iron centers, does not require the addition of reductant to promote its enzymatic activity. ... 

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