Purple acid phosphatases active sites

Kidney Bean Purple Acid Phosphatase Active Site... 

Ser/Thr-protein phosphatases are ubiquitous enzymes which constitute the catalytic domains of multiprotein complexes. They are responsible for the dephosphorylation of a range of phosphoproteins. Several protein phosphatases have been characterized by X-ray crystallography and display an active site structure similar to purple acid phosphatase. 

There are three mechanistic possibilities for catalysis by two-metal ion sites (Fig. 10). The first of these is the classic two-metal ion catalysis in which one metal plays the dominant role in activating the substrate toward nucleophilic attack, while the other metal ion furnishes the bound hydroxide as the nucleophile (Fig. 10 a). Upon substrate binding, the previously bridged hydroxide shifts to coordinate predominately with one metal ion. Enzymes believed to function through such a mechanism include a purple acid phosphatase [79], DNA polymerase I [80], inositol monophosphatase [81],fructose-1,6-bisphosphatase [82], Bam HI [83], and ribozymes [63]. 

It was clear for some time that a number of zinc enzymes required two or more metal ions for full activity, but in the absence of X-ray structural data the location of these metal centres with regard to one another was often uncertain. When the first 3-D structures began to appear, it became clear that the metals were in close proximity. A particular feature of many of these enzymes was the presence of a bridging ligand between two of the metal sites, usually an Asp residue of the protein, which is occasionally replaced by a water molecule. While some of the sites contain only Zn ions, several contain Zn in combination with Cu (in cytosolic superoxide dismutases) Fe (in purple acid phosphatases) or Mg (in alkaline phosphatase and the aminopeptidase of lens). 

The binuclear iron unit consisting of a (p,-oxo(or hydroxo))bis(p.-carboxylato)diiron core is a potential common structural feature of the active sites of hemerythrin, ribonucleotide reductase, and the purple acid phosphatases. Synthetic complexes having such a binuclear core have recently been prepared their characterization has greatly facilitated the comparison of the active sites of the various proteins. The extent of structural analogy among the different forms of the proteins is discussed in light of their spectroscopic and magnetic properties. It is clear that this binuclear core represents yet another stractural motif with the versatility to participate in different protein functions. 

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. 

The first crystal structure (with a resolution of 2.9 A) of purple acid phosphatase (kidney bean, composed of 432 amino acid residues) containing a dinuclear FeUI-Znn active site 31 has been reported (30). 

The transferrins are proteins that bind and transport iron as peIII 16-U.8 They indude lactoferrin from milk, ovotransferrin from egg white, and serum transferrin from a range of organisms. Uteroferrin, considered in Section 62.1.5.5.2 on the purple acid phosphatases, is an iron-binding protein with phosphatase activity, that has been proposed to transport iron from maternal to foetal circulation.824 826 There are distinct differences between the iron-binding sites in uteroferrin and transferrin, and so uteroferrin will not be discussed in this section. 

In nature, many enzymes that hydrolyze phosphate monoesters are activated by two or more metal ions. They include alkaline phosphatase [79], purple acid phosphatase [80], inositol monophosphatase [81], and D-fructose 1,6-biphosphate 1-phosphatase [82]. The active sites of protein serine and threonine phosphatases also consist of dinuclear... 

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). 

The active site similarities listed above belie a remarkable functional diversity, which includes phosphate ester hydrolysis, dioxygen and NO reduction, reversible O2 binding, and O2 activation, the last of which includes enzymes involved in ribonucleotide reduction, hydrocarbon monooxygenation, and fatty acyl desaturation. At the overall protein level, the purple acid phosphatases (PAPs) seem to be completely unrelated, both structurally and functionally, to any of the others in this class. Similarly, the flavo-diiron enzymes form a structurally and probably functionally distinct family of proteins, catalyzing both dioxygen and NO reduction. These last two examples illustrate that attempts to shoehorn all of these enzymes into a single class can sometimes provide a simplistic and misleading view of their chemistry and biochemistry. 

The alkaline phosphatase family contains three metals in the active site while the rest of the cocatalytic zinc sites contain two metals (Figme 10) (Table 3). Some of these sites contain metals such as copper, iron, and magnesium in combination with zinc. Combinations of Cu(II)/Zn are seen in the superoxide dismutase (SOD) family see Copper Proteins with Type 2 Sites), Zn/Mg are seen in alkaline phosphatase family and lens aminopeptidase, and Fe(III)/Zn in the purple acid phosphatase family. 

Fig. 21 The active site of kidney bean purple acid phosphatase.

Metallo-dependent acid phosphatases (a.k.a. purple acid phosphatases) contain a binuclear ion center [18, 19] in the active site which confers a characteristic purple color in solution. The color is... 

The nature of the metal-ions in the active site also varies between species. Whereas the purple acid phosphatase isolated from red kidney beans (rkbPAP) contains Fe and Zn", the tartrate-resistant acid phosphatase isolated from rat osteoclasts (TRAcP) contains two iron atoms in different oxidation states, an stabilized Fe ion and a redox-active Fe ion. In this way, the ability of the ferrous ion to act as an electron donor confers to the enzyme an alternative function as generator of reactive oxygen species (ROS) [20, 21]. The enzyme may appear in an inactive purple form when the redox-active iron is oxidized to the ferric state, or it can be in an active pink form where the redox-active iron is reduced to the ferrous state [22]. In particular, the tartrate-resistant acid phosphatase isolated from osteoclasts is synthetized as a precursor which is activated by cysteine proteinases resulting in an active two subunit enzyme [23]. 

Davis JC, Averill BA (1982) Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen. Proc Natl Acad Sci USA 79 4623-4627... 

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. 

Figure 17-3 Active site dinuclear metal centers of (A) purple acid phosphatase [29, 30], (B) PPl [31, 32], (C) calcineurin [15, 16], and (D) protein phosphatase 2C [27] adapted from the X-ray structures. Figures in A, B, and C are reproduced with permission from [18],...

---