Apase

Acid phosphatase (AP/APase) (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) catalyzes hydrolysis of phosphate monoesters in acidic condition with pH 4–7. Few members of AP family are metallohydrolase having two heterovalent metal ions in catalytic center. Most of isoforms show a characteristic purple color due to charge transfer fromtyrosine residue to Fe (III). Thus, they are called as purple acid phosphatase (PAP). These have also been given name tartrate-resistant acid phosphatases (TRAP) because of their insensitive nature to L(+)-tartrate inhibition whereas some isoforms are sensitive to tartrate inhibition. It is abundantly present in nature and has been studied in plants, animals, and lower organisms like bacteria and fungi. It has a significant role in various metabolic processes.

Distribution

AP is widely distributed in all living systems from lower to higher organisms. Different plant parts like in cell wall of Pisum sativum and Nicotiana tobaccum, phloem of N. tobaccum, and shoot of Vigna aconitifolia express AP. In animals, this has been isolated from bovine, human spleen, bone, and macrophages. Including this, prostatic AP has been characterized from prostate gland, liver, spleen, and brain. Lysosomal AP was isolated from lysosomes of mast cells. It has also been reported in fungal species. In Saccharomyces cerevisiae, it is localized in periplasmic space, cell wall, and in a few cell organelles. Some prokaryotic genomes also carry sequences homologous to human and plant PAP.

Molecular Structure

Each subunit of HMW plant PAP possesses two domains. The first one is amino-domain of unknown function which is formed by sandwiched β-sheets, and the second is the catalytic domain, i.e., carboxyl-domain. Catalytic domain is structured by mixed β sheets (α/β strands). In contrast, LMW mammalian TRAP has only one domain resembling with catalytic domain of plant PAP. Generally, PAPs consist of two metal ions making two coordination spheres with seven highly conserved amino acid ligands in active site.

It is observed that, in PAP/TRAP, among two metal ions, one is ferric ion (Fe3+) involved in charge transfer with tyrosine residue and the second is divalent metal ion which may be Fe, Zn, or Mn. Mammalian TRAP contain Fe (II) as second metal site and similarly, PAP from plant and prokaryotic origin have Zn (II) or Mn (II) as divalent metal ion. A specific pattern of seven amino acid residues involved in metal–ligand coordination are invariant among most of species from prokaryotes to eukaryotes. Thus, this unique sequence can be considered as signature sequence of PAP/TRAP. Fe (III) is coordinated with a tyrosine, a histidine, and an aspartate residue, and M (II) is coordinated with an asparagine and two histidine residues. Mostly, APs are glycoprotein with N-linked oligosaccharide chain. Some specific sites for N-glycosylation have been recognized on amino terminal of the enzyme subunits from various species.

Catalytic Mechanism

Figure 1. Catalytic mechanism proposed for PAP. (Schenk G; et al. 2008)

Since catalytic site of AP in plants, animals, and bacteria is somewhat homologous, these may follow the similar catalytic mechanism. Although the crystal structure of AP from various sources has been studied thoroughly, catalytic mechanism of this enzyme needs to be explored further. A number of hypotheses for its reaction mechanism have been proposed. According to the mechanism proposed by Klabunde et al. for red kidney bean PAP, divalent metal ion is bound to monodentate phosphate group of the substrate, and a hydroxide ion bound to Fe (III) acts as nucleophile whereas mechanism proposed for sweet potato illustrates a tripodal mode of coordination of the substrate with two metal species where heterovalent metal ions in active center are bridged to two oxygen atoms of phosphate group of the substrate, and third phosphate oxygen bridging both metal ions initiates hydrolysis. Trivalent Fe metal activates the nucleophile, i.e., metal-bound μ-hydroxo group which breaks the phosphoester bond. A comprehensive structural analysis of red kidney bean PAP gives a view on its catalytic behavior. In addition, few metal replacement studies reveal the importance of metal ions in its catalytic property. Some mutagenic and crystallographic studies describe about a repression loop in the mammalian TRAP which regulates the catalytic activity by inhibiting the formation of enzyme–substrate complex. In crystal structure of human PAP, carboxy side chain of Asp145 on this loop acts as a bidentate ligand bridging two metal ions and thus blocks catalytic site.

Applications

• Development of Stress-Tolerant Plant Varieties

Pi fertilizers come from non-renewable sources like rock phosphate and excess use of these increases eutrophication in water bodies. In order to save these nonrenewable natural sources and environment, sustainable agriculture is being now encouraged. Genetic engineering in plants is one of the revolutionary approaches to improve crop varieties. By using this, Pi starvation inducible genes can be introduced into plants to overcome phosphorus deprivation. To date, different isoforms of Pi-starvation-inducible PAP from various plant sources like A. thaliana, N. tobaccum, and P. vulgaris have been isolated and characterized in detail.

• TRAP in Clinical Diagnosis and Prognosis

In human, TRAP isoforms 5a and 5b are distributed in various tissues. Dimeric TRAP 5b is basically expressed in osteoclasts whereas TRAP 5a is produced by immune cells like macrophages and dendritic cells associated with pathogen processing. Both isoforms could be used as markers for chronic inflammation in different organs and bone-related disorders like osteoporosis, bone metastases in cancer, etc., respectively.

• Immobilized AP

The most important use of immobilized enzyme is construction of various types of biosensing devices. These sensors were very useful in detection of environmental pollutants and heavy metals. An amperometric biosensor by using AP and polyphenol oxidase has been constructed to detect As (V) contamination in water.

References

1. Anand A; Srivastava P K. A molecular description of acid phosphatase. Applied Biochemistry & Biotechnology, 2012, 167(8):2174-2197.
2. Schenk G; et al. Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle. Bmc Structural Biology, 2008, 8(1):6.