Purple acid phosphatases comprise a family of binuclear metal-containing acid hydrolases, representatives of which have been found in animals, plants, and fungi. The goal of this study was to characterize purple acid phosphatases from sweet potato tubers and soybean seeds and to establish their relationship with the only well-characterized plant purple acid phosphatase, the FeIII-ZnII-containing red kidney bean enzyme. Metal analysis indicated the presence in the purified sweet potato enzyme of 1.0 g-atom of iron, 0.6 -0.7 g-atom of manganese, and small amounts of zinc and copper. The soybean enzyme contained 0.8 -0.9 g-atom of iron, 0.7-0.8 g-atom of zinc per subunit, and small amounts of manganese, copper, and magnesium. Both enzymes exhibited visible absorption maxima at 550 -560 nm, with molar absorption coefficients of 3200 and 3300 M ؊1 cm ؊1 , respectively, very similar to the red kidney bean enzyme. Substrate specificities were markedly different from those of the red kidney bean enzyme. A cloning strategy was developed based on N-terminal sequences of the sweet potato and soybean enzymes and short sequences around the conserved metal ligands of the mammalian and red kidney bean enzymes. Three sequences were obtained, one from soybean and two from sweet potato. All three showed extensive sequence identity (>66%) with red kidney bean purple acid phosphatase, and all of the metal ligands were conserved. The combined results establish that these enzymes are binuclear metalloenzymes: Fe-Mn in the sweet potato enzyme and Fe-Zn in soybean. The sweet potato enzyme is the first welldefined example of an Fe-Mn binuclear center in a protein. © 1999 Academic PressKey Words: acid phosphatase; binuclear metalloenzyme; purple acid phosphatase; sweet potato; soybean.In plants the assimilation and maintenance of adequate levels of phosphate require the presence of a group of enzymes which release phosphate from phosphate esters and anhydrides (1). Depending on the optimum pH for activity these enzymes are classified as acid or alkaline phosphatases. Detailed characterization of the enzymes is necessary in order to determine their individual biological roles.Purple acid phosphatases catalyzing the hydrolysis of a broad range of phosphoric acid esters and anhydrides have been characterized from animal, plant, and fungal sources (for a recent review see Klabunde and Krebs (2)). The mammalian enzymes are closely related with respect to size (ϳ35 kDa), amino acid sequence, and metal content. The active form of the enzyme is pink ( max ϭ 510 nm) and contains a binuclear FeIII-FeII metal center in which the iron atoms are antiferromagnetically coupled. Oxidation converts the enzyme to the inactive, purple FeIII-FeIII form ( max ϭ 550 nm). Plant purple acid phosphatases have been isolated from red kidney bean (3) and soybean seeds (4, 5), sweet potato tubers (6 -8), spinach leaves (5), duckweed (9), and suspension cultures of rice (10) and soybean (11). The well-characterized red kidney bean enzyme is a homodimer, each subunit ...
Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the -barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.protein conformation ͉ GTP inhibition ͉ transamidase activity A llosteric regulation of enzymes by conformational change is an important means of biological control, involving residues that functionally couple ligand binding at the allosteric site to modification of the catalytic site. Transglutaminase type 2 (TG2), also known as tissue TG or G h (high molecular weight GTP-binding protein), is a multifunctional protein that is allosterically regulated by calcium and GTP (1). TG2 catalyzes calcium-dependent transamidation reactions, resulting in posttranslational amine modification of proteins or cross-linking of interchain glutamine and lysine residues to form N (␥-glutamyl)lysine isopeptide bonds, which confer rigidity and protease resistance on protein complexes (2). TG2 is also a GTPase (3) and mediates intracellular signaling by various G protein-coupled receptors (4-6).GDP-bound human TG2 (7) is comprised of four domains: an N-terminal -sandwich, a core domain in which the transamidase active site catalytic triad (Cys-277, His-335, and Asp-358) and transition-state stabilizing residue (Trp-241) (8) are buried and inaccessible to substrate, and two -barrels. Nucleotide binds mainly to residues from the first and last strands (amino acids 476-482 and 580-583) of -barrel 1 and to two core domain residues (Lys-173 and Phe-174) that protrude on a loop to meet -barrel 1 (7, 9, 10). This is postulated to stabilize two -barrel 1 loops that block access to the catalytic site (7). One of these loops protrudes into the core domain localizing Tyr-516 within hydrogen-bonding distance of Cys-277 (7). This is postulated to prevent Cys-277 interaction with the substrate (7,11,12). Calcium-activated TG2 has unique conformational epitopes (13) and is less compact (14-16) and less resistant to protease digestion (1, 10, 14) than GTP-bound TG2. Allosteric mechanisms governing the conformational switch between transamidase and GTPase functions have yet to be elucidated.Mutation of Arg-579 in rat TG2 (Arg-580 in ...
Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal~35 kDa, plant~55 kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.
Purple acid phosphatases comprise a family of binuclear metal-containing enzymes, the members of which have been identified in plants, animals, and fungi. The animal enzymes contain an antiferromagnetically coupled binuclear iron center. The active form of the enzyme (Fe(III)-Fe(II)) exhibits a distinctive EPR signal with principal g values of 1.93, 1.75, and 1.50 (1), whereas the inactive (Fe(III)-Fe(III)) form is EPRsilent at X-band microwave frequencies (2, 3). Plant purple acid phosphatases appear to be more diverse. Red kidney bean purple acid phosphatase, the best characterized plant enzyme, contains an Fe(III)-Zn(II) center (4). Replacement of Zn(II) by Fe(II) yields an enzyme with full activity and with spectral properties very similar to those of the animal enzymes (5, 6).The sequences of three cDNAs encoding different isoforms of sweet potato purple acid phosphatase have recently been reported (7,8). Two of these isoforms have been purified. One form was shown by Durmus et al. (7) to contain one g-atom of iron and 0.77 g-atom of zinc/subunit. Comparison of its spectroscopic properties with those of the red kidney bean enzyme indicated that the metal ions are present as a binuclear Fe(III)-Zn(II) center (7). A different form exhibiting 66% sequence identity to the Fe(III)-Zn(II) enzyme has recently been characterized in our laboratory. It contains one g-atom of iron and 0.6 -0.8 g-atom of manganese/subunit (8). We now report enzymatic, magnetic susceptibility and EPR studies on this enzyme demonstrating that the enzyme contains a catalytically active Fe(III)-Mn(II) center that is strongly antiferromagnetically coupled, providing the first reported evidence for such a center in a protein. EXPERIMENTAL PROCEDURESPurification and Enzyme Assay-The enzyme was purified by a combination of juice extraction, acetone and ammonium sulfate fractionation, DEAE-cellulose chromatography at pH 7.0, and gel filtration on a Sephadex G-150 Superfine column at pH 4.90 as described elsewhere (8). The enzyme was purple when concentrated and exhibited essentially the same visible absorption spectrum ( max ϭ 560 nm, ⑀ max ϭ 3207) as the red kidney bean enzyme ( max ϭ 560 nm, ⑀ max ϭ 3360) (4). Enzyme assays were performed at 25°C using p-nitrophenyl phosphate (5 mM) as substrate in 0.1 M acetate buffer, pH 4.90. The specific activity of the sample used for magnetic susceptibility and EPR measurements was 675 units/mg. The protein subunit concentration was 0.556 mM (based on a subunit weight of 55 kDa and an A 1% 1 cm at 280 nm of 27.03 (8)). Metal Ion Analysis-Metal ion content was determined by inductively coupled plasma mass spectrometry using a PerkinElmer SCIEX-ELAN 5000 spectrometer. Samples and standards were prepared in 0.1% HNO 3 . Separate standard curves were routinely prepared for iron, zinc, copper, and manganese. Samples were measured in quadruplicate. Metal ion analysis showed that the sample of enzyme used for magnetic susceptibility and EPR measurements contained 1.035 Ϯ 0.108 iron, 0.582 Ϯ 0.044 manganese, 0...
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