1. LITERATURE REVIEW
1.4 ACID PHOSPHATASE
1.4.3 Properties of acid phosphatase
1.4.3.1 Physical properties
Most acid phosphatases are glycoproteins and display varying molecular weight sizes, PI and subunit structure depending on the source of isolation. Resistance to inhibition by tartrate also distinguishes type 5 acid phosphatase from acid phosphatase of lysosomal (VON FIGURA and WEBER, 1978) or prostatic (VIHKO et al., 1978) origin. The animal enzymes are also distinguished from lysosomal and prostatic acid phosphatases by their resistance to inhibition by L (+) tartrate, and are commonly referred to TRAPS.
Mammalian tartrate resistant acid phosphatase (TRACP, TRAP or TR-AP) also known as
22 type 5 acid phosphatase (AcP5, E.C 3.1.3.2) (VIHKO et al., 1978). Together with similar enzymes isolated from animals, plants and fungi, it belongs to the group of purple acid phosphatases (PAP).
PAPs from mammalian sources (human, cow, pig and mouse) are reported to be monomeric with a molecular size of approximately 35 kDa. These enzymes contain an Fe (III-Fe (II) binuclear metal centre and exhibit high sequence identity (>80%) (KLABUNDE and KREBS, 1997). On the other hand, plant PAPS (red kidney bean, sweet potato and soybean) are homodimeric and are composed of a subunit molecular mass of approximately 55-110 kDa. Thus, they are generally characterized as high molecular weight acid phosphatases. These plants‟ PAPs are more diverse with respect to metal centres Fe (III)-Zn (II) or Fe (III)-Mn (II) (BECK et al., 1986; LEBANSKY et al., 1992; SCHENK et al., 1999). A number of plant genes putatively encoding low molecular weight (Mr) PAPs have been identified (SCHENK et al., 2000).
The PAP enzymes have been reported to be present in the cyanobacterium Synechocytistis sp as well as in Mycobacterium tuberculosis and M. leprae (SCHENK et al., 2000). Until recently, Aspergillus ficuum was the only fungal species reported to have a monomeric PAP with a molecular mass of approximately 85 kDa (ULLAH and CUMMINS, 1988).
Since the completion and sequencing of the genome of several Aspergillus sp, other PAPs such as Aspergillus oryzae (MACHIDA et al., 2005) and Aspergillus flavus (NCBI Reference Sequence: XP_002376348.1, deposited by NIERMAN, 2007) have been reported. Mammalian PAPS are highly conserved with 80% amino acid homology (SCHENK et al., 2000). Interestingly, low sequence identity (20%) has been found between plant and mammalian PAPs, except for the metal-ligating amino acid residues which are identical (KLABUNDE et al., 1995; SCHENK et al., 2000).
Most acid phosphatases of yeasts and fungi are glycoproteins, like most proteins that work in the extracellular milieu (Table 1.1). For instance, in yeast apases, the sugar component represents 50% of the enzyme in Saccharomyces cerevisiae (SHNYREVA et al., 1992)
23 and 40% of the enzyme in Rhodoturula glutinis (TRIMBLE et al., 1981). It has been estimated that about a third of all proteins that enter secretory pathways in eukaryotic cells may be N-glycosylated and tens of thousands of glycoprotein variants may coexist in eukaryotic cells (WALSH et al., 2005). In addition, these proteins have a tendency to form tetramers in solution. Aspergillus niger pH 2.5 acid phosphatase has an apparent native molecular mass of 269 kDa with a glycosylated subunit of approximately 65 kDa and an unglycosylated form of 50.8 kDa. It forms a tetramer in solution (KOSTREWA et al., 1999). In Penicillium funiculosun the phosphatase appear to be a 76 kDa heterodimer composed of 51 and 26 kDa subunits quantified on SDS-PAGE.
All apases (mammalian, plants and microorganisms) have pH optima below 7.0 (Table 1.1). However, the ranges vary immensely from species to species ranging from 2.5-6.0.
For example, in Aspergillus ficuum three apases with pH optima between 2.2, 2.5, and 5.5 were isolated by ULLAH and CUMMINS (1987). Some apases have a wide pH optima, for example the apase found in A. fumigatus, is active at pH 3.0 to 7.0, with the optimum activity occurring between pH 4.0 and 6.0 (BERNARD et al., 2002).
The conserved active site (N-terminal) RHGXRXP, (where X represents any amino acid) is a hallmark feature of all high molecular acid phosphatases as well as R and HD motifs located at almost identical positions in the active sites (OSTANIN et al., 1992;
SCHNEIDER et al., 1993; KOSTREWA et al., 1997; CHI et al., 1999; MULLANEY et al., 2000) (Table 1.2). In contrast, low molecular apases are devoid of this motif.
24 Table 1.1: Physical properties of various acid phosphatases, isolated from different organisms. T= Optimal temperature. PI= isoelectric point.
Sources Protein‘s nameForms Native Mr (kDa)
PI pH T (°C) Glycoprotein References MAMMALIAN:
Homo sapiens TRACP I 174.40 3.8-4.1 4.5 - yes ROBINSON and GLEW, 1980
Rat PAP II 150 - - yes VIHKO et al., 1993
Homo sapiens TRACP I 35-37 - 2.5 - yes VIHKO et al., 1978
PLANTS : Tomatoes (Lycopersicon esculentum
6-phyt - 4.3 - 4.3 - yes LI et al., 1997
Potato Apase II 100 - 5.8 - yes GELLATLY et al., 1994
Wheat Apase III 55 - 4.5-5.5 - yes VERJEE, 1969
BACTERIA:
Escherichia coli Phyt II 44.7 6.3-6.5 4.5 60 yes GOLOVAN et al., 2000
Bacillus subtilis (natto) Phytase - 36-38 - 6.0-6.5 60 - SHIMIZU, 1992
FUNGI & YEAST:
Pichia pastoris apase - - - yes HAN and LEI, 1999
Saccharomyces cerevisiae
Phyt I 100 2.0-2.5 3.5 55-60 yes HAN et al., 1999
Aspergillus niger 3Phyates I 100 5.0 5.0 - yes DVOŘÁKOVÁ et al., 1997
25 Table 1.2: Amino acid sequence alignments of the conserved region where acid phosphatase from different organisms share a high degree of amino acid conservation “RHGXRXP”. PAP= purple acid phosphatases.
Organisms Acc. No Sequences protein Location References
Homo sapiens NP_001601 RFVTLLYRHGDRSPVK PAP Lysosomal POHLMANN et al., 1988; WANG et
al., 2008
Rattus norvegicus P20611 RFVTLLYRHGDRSPVK- PPAL Lysosomal HIMENO et al ., 1989
Aspergillus niger A2QSK3 DQVIMIKRHGERYPSP 3-phytase B Cell membrane PEL et al., 2007
Saccharomyces cerevisiae P24031 KQLQMLARHGERYPTY PHO3 Cell membrane BAJWA et al., 1984
Schizosaccharomyces pombe CAB68657 KQVHTLQRHGSRNPTG apase Vacuole ELLIOTT et al., 1986
Bacillus cereus Q633T0 RFVTLLYRHGDSRSPVK PPA Periplasmic space HAN et al., 2006
26 The high molecular weight (histidine) acid phosphatase family is functionally conserved from prokaryotes to higher eukaryotes (CHI et al., 1999). Thus, several members of this enzyme family can be found in bacteria such as Bacillus cereus (HAN et al., 2006); fungi such as Aspergillus ficuum and A. niger (KOSTREWA et al., 1997; PEL et al., 2007) as well as in rats (HIMENO et al., 1989; SCHNEIDER et al., 1993) and the human prostate (POHLMANN et al., 1988) (Table 1.2).