Kidney bean purple acid phosphatase (KBPAP) is an Fe(III)-Zn(II) metalloenzyme resembling the mammalian Fe(III)-Fe(II) purple acid phosphatases. The structure of the homodimeric 111-kilodalton KBPAP was determined at a resolution of 2.9 angstroms. The enzyme contains two domains in each subunit. The active site is located in the carboxyl-terminal domain at the carboxy end of two sandwiched beta alpha beta alpha beta motifs. The two metal ions are 3.1 angstroms apart and bridged monodentately by Asp164. The iron is further coordinated by Tyr167, His325, and Asp135, and the zinc by His286, His323, and Asn201. The active-site structure is consistent with previous proposals regarding the mechanism of phosphate ester hydrolysis involving nucleophilic attack on the phosphate group by an Fe(III)-coordinated hydroxide ion.
Our understanding of the regulation of vascular tone has been extended since the identification of vasoactive agents such as the atrial natriuretic peptides, endothelial-derived relaxing factor and endothelin. Unidentified vasopressive agents have been found in platelets. Here we isolate these vasopressors and identify them as diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) by chromatography, mass spectrometry, ultraviolet spectroscopy and enzymatic cleavage. In the vasculature of isolated perfused rat kidney, both diadenosine phosphates were active at a concentration of 10(-9) M; in aortic rings, contractions were elicited at 10(-8) M. Intra-aortic injection in the rat caused a prolonged increase in blood pressure. We conclude that AP5A and AP6A may play a part in local vasoregulation and possibly in the regulation of blood pressure.
Oxidation of the reduced (pink) phosphate-free bovine spleen acid phosphatase with 1.5 mol HzOz or sodium peroxodisulfate/mol, in the presence of Mes or Bistris pH 5, leads to a species with an absorption maximum at 558 nm. Addition of acetate or oxidation in the presence of acetate buffer engenders a species with a maximum at 550 nm. Addition of phosphate to both species shifts the maximum immediately to 540 nm; this is the species also found after preparation from the spleen. The assumption that these species represent strongly bidentatebinding hydroxo, acetato and phosphato complexes of the Fe(II1)-Fe(II1) system is supported by replacement reactions with other ligating oxoanions followed by their typical spectral shifts. These oxoanion complexes cannot be dissociated by gel filtration; this is possible only after reduction to the Fe(I1)-Fe(II1) system. The oxidized species without EPR signals below g values of 2 still reveals 5% activity which cannot be reduced to zero even in the presence of higher concentrations of peroxodisulfate. The pH optimum of the reaction with a-naphthyl phosphate shifts from 5.9 to 5.3 in the oxidized species.The apparent pK values around 4.5 as derived from the pH dependence of activity, of the EPR spectra, and the spectral shifts of the phosphate-saturated reduced and oxidized species are assigned to an aquo/hydroxo equilibrium at the Fe(Il1) or an equilibrium, where the phosphato ligand is replaced by a hydroxo ligand. A reaction mechanism is proposed in which a hydroxo ligand at the chromophoric Fe(II1) attacks the phosphoric acid ester group only when that is monoprotonated and pre-oriented by electrostatic interaction with the nonchromophoric metal ion. Binding and inhibition studies with the oxoanions indicate that they compete with the catalytically active hydroxo group of the reduced and oxidized enzyme with nearly the same inhibition constants. Catalysis is not affected by the oxoanions which replace the additional p-hydroxo ligand in the 558-nmabsorbing Fe(II1)-Fe(II1) species. In contrast to hemerythrin and ribonucleotide reductase, a binuclear iron center is proposed for the purple acid phosphatase, which is bridged by a carboxylato and two aquo/hydroxo groups, but without a p-0x0 bridge.Mammalian purple tartrate-resistant acid phosphatases (PAP) [l, 21 have been isolated from porcine uterus [3], bovine spleen [4, 51, rat spleen [6] and rat bone [7]. They are glycoproteins [4-81 with molecular masses of 35 kDa with a monomeric peptide structure. The chain of the bovine enzyme seems to be split into two nonidentical subunits [5]
both metals. Other diiron proteins, like hemerythrin, ribonucleAbstract The primary structure of nteroferrin (Uf), a 35 kDa otide reductase, and methane monooxygenase, involved in monomeric mammalian purple acid phosphatase (PAP) containtransportation and activation of dioxygen, share similar core ing a Fe(llI}--Fe(lI) center, has been compared with the sequence units, as revealed by their X-ray structures [13][14][15]. An antiparof the homodimeric 111 kDa Fe(llI)-Zn(II) kidney bean purple acid phosphatase (KBPAP). The alignment suggests that the allel four-helix-bundle is a common tertiary structure motif for amino acid residues ligating the dimetal center are identical in Uf these diiron proteins. and KBPAP, although the geometry of the coordination sphereThe intensively studied plant purple acid phosphatase from might slightly differ. The recently determined X-ray structure of KBPAP [19] reactivated phosphoric acid esters and anhydrides in the pH vealed the geometry of the catalytic Fe(III)-Zn(II) center. A range from 4 to 7. They have in common a dinuclear metal schematic view is shown in Fig. 1. Fe(III) is coordinated by center with a tyrosine ~ Fe(III) charge transfer transition reTyr 167, by the N~ of His -~25, and by a monodentate carboxylate, sponsible for their characteristic purple colour and differ from Asp Z35. Zn(II) is ligated by the N, of His 's6, the N6 of His 323 and other acid phosphatases in their insensitivity to tartrate inhibithe carboxamide oxygen of Ash 2°~. The two metal ions are tion (for review see [1][2][3]).bridged by the monodentate carboxylate group of Asp a64. In PAPs obtained from mammalian sources, including porcine agreement with kinetic and spectroscopic data a/l-hydroxo uterine fluid (uteroferrin, Uf), bovine spleen, macrophages, and bridge and two terminal solvent molecules, an aqua ligand at osteoclasts are monomeric Fe(lI1)-Fe(II) proteins of approxiZn(II) and a hydroxo ligand at Fe(III), were modeled to commately 35 kDa. In addition to the hydrolytic function a role in plete the coordination sphere of the Fe(III)-Zn(II) center. the activation of dioxygen has been discussed for the mammaThus, the ligands revealed by ~H NMR for the mammalian lian PAPs [4,5]. An X-ray structure of one of the mammalian PAPs [6] are also present in the Fe(III)-Zn(II) plant enzyme. PAPs is not yet available. NMR studies reveal the ligation of Some differences in the redox behaviour and in the visible one tyrosine and one Ne-coordinated histidine to iron(Ill) and spectra, however, indicate that at least the geometry of the the ligation of one N6-coordinated histidine to iron(II) in uteractive sites cannot be exactly identical. oferrin and the bovine spleen enzyme [6]. Definitive informaNo similarity in the amino acid composition and only very tion about the remaining protein ligands could not be obtained, low homology in the sequence (20% identity) could be observed although further paramagnetically shifted proton signals indibetween the kidney bean enyzme and the mammalian proteins. cate the ...
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