Purification, enzymatic properties, and active site environment of a novel manganese(III)-containing acid phosphatase.
A new manganese-containing acid phosphatase has been isolated and crystallized from sweet potato tubers. The pure enzyme contains one atom of manganese per Mr = 110,000 polypeptide and shows phosphatase activity toward various phosphate substrates. The pH optimum of the enzyme was 5.8 and the enzyme activity was inhibited by Cu2+, Zn2+, Hg2+, AsO43-, and MoO42-. This stable metalloenzyme is red-violet in color with an intense absorption band at 515 nm (epsilon - 2460). Our electronic, circular dichroism, and electron spin resonance findings strongly indicate that the Mn-valence state of the native enzyme is trivalent. When the Mn-enzyme is excited by the 5145 A line of Ar+ laser, prominent Raman lines at 1230, 1298, 1508, and 1620 cm-1 were detected. This Raman spectrum can probably be interpreted in terms of internal vibration of a coordinated tyrosine phenolate anion. The tryptophan-modified enzyme showed a positive Raman band at 370 cm-1, which is preferentially assigned to a Mn(III)-S streching mode. The modification of the Mn-enzyme by N-bromosuccinimide led to a large decrease in the fluorescence intensity of 335 nm which was dominated by its tryptophan residues within a considerable hydrophobic environment. The acid phosphatase activity was significantly decreased by the tryptophan modification. With respect to the active site donor sets, the Mn(III)-containing acid phosphatase is distinctly different from the Zn(II)-containing alkaline phosphatase. Of interest is also the appreciable similarity of some enzymatic and spectroscopic properties between the present enzyme and uteroferrin.
D000135:Acid Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000838:Anions; D001665:Binding Sites; D002412:Cations; D002942:Circular Dichroism; D003460:Crystallization; D007700:Kinetics; D008345:Manganese / Q000032:analysis*; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman
D000596:Amino Acids; D000838:Anions; D002412:Cations; D008345:Manganese; D000135:Acid Phosphatase