Jetten M S, Fluit T J, Stams A J, Zehnder A J
Department of Microbiology, Wageningen Agricultural University, The Netherlands.
Arch Microbiol. 1992;157(3):284-9. doi: 10.1007/BF00245163.
An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homogeneity. The molecular mass estimated by gelfiltration was 139 +/- 7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an alpha 2 beta 2 oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri- and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg2+ was required for activity. The pH optimum was 8 at 1 mM PPi and 5 mM Mg2+. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent Km for PPi of 0.1 mM in the presence of 5 mM Mg2+. The Vmax was 590 mumol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a Kcat of 1400 per second. The enzyme was found in the soluble enzyme fraction after ultracentrifugation, when cells were disrupted by French Press. Upto 5% of the pyrophosphatase was associated with the membrane fraction, when gentle lysis procedure were applied.
从索氏甲烷丝菌中分离出一种无机焦磷酸酶[E.C. 3.6.1.1]。通过三个步骤，该酶被纯化了400倍，达到表观均一性。凝胶过滤法估计其分子量为139±7 kDa。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳表明，该酶由分子量分别为35 kDa和33 kDa的亚基组成，呈α2β2寡聚体结构。该酶催化无机焦磷酸、三聚磷酸和四聚磷酸的水解，但对多种其他磷酸酯无活性。活性需要阳离子Mg2+。在1 mM焦磷酸和5 mM Mg2+存在下，最适pH为8。该酶热稳定，对分子氧不敏感，不受氟化物抑制。动力学性质分析表明，在5 mM Mg2+存在下，焦磷酸的表观Km为0.1 mM。Vmax为每分钟每毫克蛋白质水解590 μmol焦磷酸，相当于每秒1400的催化常数。当细胞通过法国压榨机破碎后，在超速离心后的可溶性酶部分中发现了该酶。当采用温和的裂解程序时，高达5%的焦磷酸酶与膜部分相关。
