Acid phosphatase purple

NATASA MITIC, GERHARD SCHENK AND GRAEME R. HANSON 

For PAPs and related binuclear metallohydrolases, a point of contention has been the number of water/hydroxide molecules coordinating to the resting active 

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Dobias B (1984) Surfactant Adsorption on Minerals Related to Flotation. 56 91-147 Doi K, Antanaitis BC, Aisen P (1988) The Binuclear Iron Centres of Uteroferrin and the Purple Acid Phosphatases. 70 1-26 Domcke W, see Bradshaw AM (1975) 24 133-170 Dophin D, see Morgan B (1987) 64 115-204... 

Corrano and co-workers have characterized phosphate ester species with a mixed donor heteroscorpionate tripod zinc species 452 Krebs and co-workers synthesized purple acid phosphatase mimics including a structurally characterized FemZnn phosphate bridged dimeric species.453... 

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. 

Figure 3. Reaction mechanism proposed for purple acid phosphatase.

Even more interesting is the observed regioselectivity of 37 its reaction with 2, 3 -cCMP and 2, 3 -cUMP resulted in formation of more than 90% of 2 -phosphate (3 -OH) isomer. The postulated mechanisms for 37 consists of a double Lewis-acid activation, while the metal-bound hydroxide and water act as nucleophilic catalyst and general acid, respectively (see 39). The substrate-ligand interaction probably favors only one of the depicted substrate orientations, which may be responsible for the observed regioselectivity. Complex 38 may operate in a similar way but with single Lewis-acid activation, which would explain the lower bimetallic cooperativity and the lack of regioselectivity. Both proposed mechanisms show similarities to that of the native phospho-monoesterases (37 protein phosphatase 1 and fructose 1,6-diphosphatase, 38 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). 

Finally we should briefly mention the purple acid phosphatases, which, unlike the alkaline phosphatases, are able to hydrolyse phosphate esters at acid pH values. Their purple colour is associated with a Tyr to Fe(III) charge transfer band. The mammalian purple acid phosphatase is a dinuclear Fe(II)-Fe(III) enzyme, whereas the dinuclear site in kidney bean purple acid phosphatase (Figure 12.13) has a Zn(II), Fe(III) centre with bridging hydroxide and Asp ligands. It is postulated that the iron centre has a terminal hydroxide ligand, whereas the zinc has an aqua ligand. We do not discuss the mechanism here, but it must be different from the alkaline phosphatase because the reaction proceeds with inversion of configuration at phosphorus. 

Figure 12.13 Coordination of the dinuclear site in kidney bean purple acid phosphatase. (Reprinted with permission from Parkin, 2004. Copyright (2004) American Chemical Society.)...

Purple acid phosphatases (PAPs) occur widely in nature and are responsible for hydrolysis of orthophosphate monoethers to alcohols under acidic conditions according to the reaction... 

Figure 3. Resonance Raman spectrum of purple acid phosphatase. Protein (5 mM) maintained at 5 C In a glass Dewar and probed with 514.5 nm excitation (within the 560 nm phenolate + Fe(III) CT band, e = 4,000 cm" M The broad, underlying feature from 400-550 cm"1 Is due to Raman scattering from glass. (Reproduced from Ref. 14. Copyright 1987 American Chemical Society.)...

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. 

Figure 1. Characteristic EPR signals of Fe(II)Fe(III) sites in semimethemerythrinj (a), semimethemerythrinQ (b), reduced uteroferrin (c), reduced uteroferrin-molybdate complex (d), reduced bovine spleen purple acid phosphatase (e), reduced component A of methane monooxygenase (f). (Reproduced with permission from ref. 26. Copyright 1987 Elsevier.)...

To what extent are the binuclear units in the purple acid phosphatases analogous to those found in hemerythrin and ribonucleotide reductase There are similarities and differences. The oxidized form is purple and EPR silent with strong antiferromagnetic coupling between the two Fe(III) centers the reduced form is pink and EPR active (gav = 1.7-1.8) with weak antiferromagnetic coupling between the Fe(III)-Fe(II) centers (3,72,73). 

A new class of metalloprotelns containing polynuclear, non-heme oxo-bridged iron complexes has emerged recently. Dinuclear centers are present in hemerythrin (Hr), ribonucleotide reductase (RR), purple acid phosphatases (PAP) and, possibly, methane monooxygenase (MMO) these centers as well as model compounds are reviewed in Chapter 8. 

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. 

Additional binuclear octahedral iron proteins have been identified. Of these the purple acid phosphatase has been the most vigorously studied and has a distinctly different biochemical function. Indeed, the protein may be involved with iron transport as well as a hydrolase (Doi et aL, 1988 Nuttleman and Roberts, 1990). Because there are insufficient structural data to provide a basis of comparison, we have omitted it from our discussion. Likewise, rubrerythrin has only recendy been identified as a member of the class, and no enzymology has been established (LeGall et al., 1988) thus, its inclusion in this discussion would be premature. There... 

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