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首页> 外文期刊>Biochemistry >The activity of oxidized bovine spleen purple acid phosphatase is due to an Fe(III)Zn(II) 'impurity'
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The activity of oxidized bovine spleen purple acid phosphatase is due to an Fe(III)Zn(II) 'impurity'

机译：氧化的牛脾紫色酸性磷酸酶的活性是由于Fe（III）Zn（II）的“杂质”

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Bovine spleen purple acid phosphatase (BSPAP) is a dinuclear iron protein with two stable redox states. The Fe3+Fe2+ state is the active state, while the fully oxidized protein (BSPAP,,) has been reported to retain 5-10% activity, corresponding to a k(cat) of ca. 150 s(-1) [Dietrich, M., Munstermann, D., Suerbaum, H., and Witzel, H. (1991) fur. J. Biochem. 199, 105-113]. Here we show that this activity does not originate from Fe3+Fe3+-BSPAP, but rather from an 'impurity' of FeZn-BSPAP. The FeZn form of BSPAP was prepared from apo-BSPAP following a new procedure, and its kinetic properties were carefully determined for comparison to those of BSPAP(ox). For the hydrolysis of p-NPP at pH 6.00, both k(cat) and KM were affected by the Fe2+-to-Zn2+-substitution [Fe3+Fe2+-BSPAP, k(cat) = (1.8 +/- 0.1) x 10(3) s(-1) and K-M = 1.2 +/- 0.2 mM; Fe3+Zn2+-BSPAP; k(cat) = (2.8 +/- 0.2) x 10(3) s(-1) and K-M = 3.3 +/- 0.4 mM]. The K-M of BSPAP(ox) was the same as that of FeZn-BSPAP. pH profiles of BSPAP(ox) and FeZn-BSPAP were both shifted to lower pH compared to that of BSPAP(red). FeZn-BSPAP, FeZn-BSPAP . PO4, and FeZn-BSPAP . MoO4 all showed characteristic EPR spectra similar to the corresponding complexes of FeZn-Uf. The same species could also be observed in concentrated samples of native BSPAP. Spin integration of these spectra showed a quantitative relation between the spin concentration of the FeZn-BSPAP 'impurity' and the residual phosphatase activity after oxidation. Since all activity found after oxidation of BSPAP could be attributed to FeZn-BSPAP, there is no direct evidence that Fe(3+)Fe(3+-)BSPAP is catalytically active. These results set an upper limit to the possible catalytic activity of the Fe3+Fe3+ form of less than or equal to 1% of that of the Fe3+Fe2+ form, a finding that is important for understanding the fundamental chemistry by which these dinuclear enzymes catalyze the hydrolysis of phosphate esters. [References: 60]
机译：牛脾紫色酸性磷酸酶（BSPAP）是具有两个稳定氧化还原状态的双核铁蛋白。 Fe3 + Fe2 +状态为活性状态，而据报道完全氧化的蛋白质（BSPAP ,,）保留5-10％的活性，相当于大约k（cat）。 150 s（-1）[Dietrich，M.，Munstermann，D.，Suerbaum，H.和Witzel，H.（1991）。 J.生物化学。 199，105-113]。在这里，我们表明该活性不是源自Fe3 + Fe3 + -BSPAP，而是源自FeZn-BSPAP的“杂质”。遵循新程序，由apo-BSPAP制备BSPAP的FeZn形式，并仔细确定其动力学性质，以与BSPAP（ox）进行比较。对于pH值为6.00的p-NPP的水解，k（cat）和KM都受Fe2 + -Zn2 +-取代[Fe3 + Fe2 + -BSPAP，k（cat）=（1.8 +/- 0.1）x 10 （3）s（-1），KM = 1.2 +/- 0.2 mM; Fe3 + Zn2 + -BSPAP; k（cat）=（2.8 +/- 0.2）x 10（3）s（-1），K-M = 3.3 +/- 0.4 mM]。 BSPAP（ox）的K-M与FeZn-BSPAP的K-M相同。与BSPAP（red）相比，BSPAP（ox）和FeZn-BSPAP的pH值均移至较低的pH值。 FeZn-BSPAP，FeZn-BSPAP。 PO4和FeZn-BSPAP。 MoO4均显示出特征性的EPR谱，类似于FeZn-Uf的相应络合物。在天然BSPAP浓缩样品中也可以观察到相同的物种。这些光谱的自旋积分表明，FeZn-BSPAP“杂质”的自旋浓度与氧化后残留的磷酸酶活性之间存在定量关系。由于BSPAP氧化后发现的所有活性都可以归因于FeZn-BSPAP，因此没有直接证据表明Fe（3+）Fe（3 +-）BSPAP具有催化活性。这些结果为Fe3 + Fe3 +形式的可能的催化活性设定了一个上限，该上限小于或等于Fe3 + Fe2 +形式的可能的催化活性，这一发现对于理解这些双核酶催化的基本化学反应非常重要。磷酸酯的水解。 [参考：60]

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