Itoh N, Morinaga N, Kouzai T
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Fukui University, Japan.
Biochim Biophys Acta. 1994 Aug 17;1207(2):208-16. doi: 10.1016/0167-4838(94)00053-0.
A novel bromoperoxidase was purified to homogeneity from the bacterium Pseudomonas putida IF-3 strain, which produces the antibiotic pyrrolnitrin. The enzyme had a molecular mass of 68,000 and was composed of two identical subunits (33,000). It was specific for I- and Br- and inactive toward Cl- and F- in the monochlorodimedone assay system. The optimum pH of the enzyme was around 4.2 and it rapidly lost its activity below 3.5, but it was stable over of range pH of 4 to 11. The purified enzyme was activated several fold by incubation with only cobalt ions, and did not contain an organic prosthetic group such as heme, flavin and cobalamin. Analyses of prosthetic metal compounds in the enzyme using plasma atomic emission spectroscopy (ICP-AES) combined with mass-spectroscopy (MS) and trace metal determination by high performance liquid chromatography (HPLC)-spectrometry, revealed that the enzyme contained 0.35 +/- 0.1 mol of cobalt ions, 1.0 +/- 0.2 mol of nickel ions, 0.8 +/- 0.2 mol of zinc ions and 2.0 +/- 0.2 mol of ferric iron per mol of enzyme, assuming the molecular weight of 68,000. There was no trace of vanadium in the enzyme, unlike in some nonheme haloperoxidases. Thus the bromoperoxidase of P. putida is a novel nonheme metal-containing bromoperoxidase.
从可产生抗生素硝吡咯菌素的恶臭假单胞菌IF-3菌株中纯化出一种新型溴过氧化物酶，使其达到同质。该酶的分子量为68,000，由两个相同的亚基（33,000）组成。在一氯二甲基酮检测系统中，它对I⁻和Br⁻具有特异性，而对Cl⁻和F⁻无活性。该酶的最适pH约为4.2，在pH低于3.5时会迅速失去活性，但在pH 4至11的范围内稳定。仅与钴离子孵育就能使纯化后的酶活性提高几倍，且该酶不含血红素、黄素和钴胺素等有机辅基。使用等离子体原子发射光谱法（ICP-AES）结合质谱法（MS）对酶中的辅基金属化合物进行分析，并通过高效液相色谱法（HPLC）-光谱法测定痕量金属，结果表明，假设分子量为68,000，每摩尔酶含有0.35±0.1摩尔钴离子、1.0±0.2摩尔镍离子、0.8±0.2摩尔锌离子和2.0±0.2摩尔铁离子。与一些非血红素卤过氧化物酶不同，该酶中没有钒的痕迹。因此，恶臭假单胞菌的溴过氧化物酶是一种新型的含非血红素金属的溴过氧化物酶。
