Purple acid phosphatases mechanism

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]. 

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. 

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). 

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). 

Little is known about the mechanism of action of the purple acid phosphatase. The beef spleen enzyme as isolated contains one tightly bound phosphate,821 but it is not certain whether this corresponds to a phosphorylated amino acid residue as found for other phosphatases. Addition of phosphate causes a shift in the visible spectrum of the enzyme. 

Alkaline phosphatases form a well-known class of proteins that perform quite interesting and complicated reactions. As previously reported, Zn enzymes, like carboxypeptidases, thermolysin, and carbonic anhydrases, consist of only one Zn atom per active center. Most of the alkaline phosphatases consist of two 96-kDa subunits, each containing two Zn and one Mg ion. The alkaline phosphatase from E. coli has been crystallized and described in full detail [4], and a mechanism has been proposed. Several enzymes in this category have been mentioned in recent years, some of them also containing different metal ions, such as iron and zinc, as in the purple acid phosphatase [5], It is likely that the detailed structure and mechanism of many more examples of enzymes that remove or add phosphate groups to proteins will become available in the next decade. 

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 reader is referred to the following sources of further information on the specific cocatalytic enzymes superoxide dismutase, and the reduced form, alkahne phosphatases, nuclease Pl, purple acid phosphatase, amidohydrolase, leucine amtnopeptidase, general comments on the mechanisms of the phosphatases and aminopeptidases, and other cocatalytic zinc enzymes. ... 

Further mechanistic studies during the last years focused on purple acid phosphatases and proposed a more detailed mechanism with up to eight steps [32]. 

Stereochemistry is another powerful tool for determining the net reaction pathway of phosphatases and sulfatases. These enzymes catalyze the net transfer of a phosphoryl or sulfuryl group to water from a monoester, producing inorganic phosphate or sulfate. Inversion results when the reaction occurs in a single step (Scheme 2, pathway a). Phosphatases that transfer the phosphoryl group directly to water with inversion typically possess a binuclear metal center and the nucleophile is a metal-coordinated hydroxide. Examples of phosphatases that follow this mechanism are the purple acid phosphatases (PAPs) and the serine/threonine phosphatases (described in Sections 8.09.4.3 and 8.09.4.4.1). Net retention of stereochemistry occurs when a phosphorylated or sulfiirylated enzyme intermediate is on the catalytic pathway, which is hydrolyzed by the nucleophilic addition of water in a subsequent step (Scheme 2, pathway b). 

The two-metal mechanisms have been known for most phosphotransferases e.g., alkaline phosphatase (7), inositol monophosphatase (35), serine/threonine phosphatase-1 (36), and purple acid phosphatase (30). The catalytic function of the metals in these multinuclear metallo-enzymes may be rather electrostatic and seems insensitive to the nature of the metal ions. 

Figure 14 Catalytic mechanisms proposed for the purple acid phosphatases (1) attack of a terminal hydroxide on the Fe on a monodentate phosphate ester substrate coordinated to the divalent metal site (2) attack of the bridging hydroxide on a bridging phosphate ester (3) attack of a hydroxide ion generated in the second coordination sphere of the Fe on a monodentate phosphate ester.

Figure 37. Proposed mechanism of hydrolysis by purple acid phosphatases. Reprinted with permission from [527]. Copyright 2006, American Chemical Society.

Klabunde T, Strater N, Frdhlich R, Witzel H, Krebs B. 1996. Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structures. J Mol Biol 259 737-748. Lindqvist Y, Johansson E, Kaija H, Vihko P, Schneider G. 1999. Three-dimensional structure of a mammalian purple acid phosphatase at 2.2 A resolution with a t-(hydr)oxo bridged di-iron center. JAfo/ Biol 291 135-147. 

Wynne CJ, Hamilton SE, Dionysius DA, Beck JL, De Jersey J. 1995. Studies on the catalytic mechanism of pig purple acid-phosphatase. Arch Biochem Biophys 319 133-141. 

Elliott TW, Mitic N, Gahan LR, Guddat LW, Schenk G. 2006. Inhibition studies of purple acid phosphatases implications for the catal34ic mechanism. J Braz Chem Soc 17 1558-1565. 

Aquino MAS, Lim JS, Sykes AG. 1994. Mechanism of the reaction of different phosphates with the iron(II)iron(in) form of purple acid phosphatase from porcine uteri... 

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