Environment of Mn(III) and its function in a red-violet colored acid phosphatase

Abstract

Purple colored metal-containing acid phosphatases have been purified from beef spleen, uterine fluid, sweet potato, soybean, and spinach leaves [1–3]. These acid phosphatases are divided into subgroups of Mn-containing acid phosphatases from some plants and Fe-containing acid phosphatases from mammalian organs. We have isolated and crystallized Kintoki sweet potato tubers [4] Atomic absorption analyses indicated the presence of one Mn ion per one mol of enzyme molecule (MW 110,000). This red-violet Mn-containing acid phosphatase gave an intense absorption maximum at 515 nm (ϵ = 2460) and a CD extreme at 555 nm (Δϵ = −0.53), respectively. The ratio of Δϵ/ϵ (Kuhn's anisotropic factor) was 2 × 10 −4 for the characteristic visible band. The value is typical of an electrically allowed charge-transfer transition. No ESR signals were detected in the native enzyme at 293 and 77 K. In contrast, the acid- and heat-treated colorless enzyme showed characteristics six-line ESR patterns, around g = 2, based on the aquated Mn(II) ion (I = 5/2). The results is reasonable if it is assumed that the violet chromophore represents as ESR-silent form of the metal, Mn(III). In addition this enzyme showed the absorption band at 1160 nm, which can be assigned to a transition 5A– 5B of high-spin Mn(III). The intense 515 nm band was also assigned to an electrically allowed charge-transfer band from the ligand to the metal, which was expected in Mn(III)(d 4) rather than Mn(II)(d 5). The resonance Raman spectrum of the native enzyme, excited by the 5145 Å line of argon ion laser, exhibited prominent Raman lines at 1230, 1289, 1508, and 1620 cm −1. Similar resonance Raman lines have been reported in Mn(III)-ovotransferrin (1173, 1264, 1501, and 1603 cm −1), Fe(III)-transferrin (1174, 1264, 1508, and 1613 cm −1) and uteroferrin (1177, 1265, 1505, and 1607 cm −1). In these Mn(III)- and Fe(III)-proteins to the four characteristic Raman lines have been assigned to the vibration of the coordinated phenolate anion. Accordingly, the resonance Raman spectrum of the Mn(III)-containing acid phosphatase is interpreted in terms of the internal vibrations of a coordinated phenolate anion [5]. In the shorter wave number region (<1000 cm −1), the resonance Raman spectra of the native enzyme were obscured by fluorescent background, which is due to the tryptophan residues. Tryptophan modification of the enzyme by N-bromosuccinimide showed a marked decrease of fluorescence and the N-bromosuccinimide-treated enzyme exhibited a positive resonance Raman band at 370 cm −1. Such a Mn(III)S stretching mode at approximately 370 cm −1 was reported in the infrared spectrum of the tris(N,N-diethyl dithiocarbamato) Mn(III) complex. Symmetric stretching vibrations of sulfhydryl sulfur to Fe(III) bonds have been assigned at 315–365 cm −1 in iron sulfur proteins and synthetic ironsulfur clusters. Therefore, the resonance Raman line at 370 cm −1 is assigned to Mn(III)z.sbnd;S(cysteine) stretching mode [6]. In the Mn(III)-containing acid phosphatase the enzyme activity was reduced in parallel with a decrease in the 515 nm absorption attributed to the Mn ion directly coordinated with some amino acid residues. Mn(III)-chelating reagents strongly inhibited the phosphatase activity and decreased PRR (proton relaxation rate of water) enhancement of enzyme solution. 31P NMR spectra of the enzyme in the presence of phosphate as substrate showed the pronounced broadening (full width at half-height: 37.8 ppm) 31P phosphate resonance line. The broadening effect corresponded well to 31P NMR signal of the model pyrophosphateMn(III) complex (full width at half-height: 36.2 ppm). Addition of phosphate to this enzyme solution decreased PRR enhancement by 25%. These evidences indicated strongly that Mn(III) is located on the active center in the enzyme, binds to phosphate esters, and plays an essential role in the catalytic reaction of hydrolysis of phosphomonoesters. In conclusion, (1) only single Mn ion per one enzyme molecule is contained in the enzyme, (2) the trivalenT Mn is coordinated by tyrosine and cysteine residues of the protein molecule, and (3) phosphate binds to the Mn(III) active center of this enzyme. Acid phosphatase uses Mn(III) ion to induce effective binding of phosphate substrate in an acidic environment. In our latest experimental results we note similarities and differences between plant's Mn(III)- and mammalian Fe(III)-containing acid phosphatases.