A role for iron in an ancient carbonic anhydrase
A role for iron in an ancient carbonic anhydrase
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DOI：
10.1074/jbc.m311648200
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发表时间：
2004-02-20
影响因子：
4.8
通讯作者：
Ferry, JG
中科院分区：
文献类型：
--
作者：
Tripp, BC;Bell, CB;Ferry, JG
自1933年以来，碳酸酐酶的研究主要集中在哺乳动物(α类)和植物(β类)的酶上；然而，最近又发现了两个额外的类(伽马类和德尔塔类)。CaM来自嗜热甲烷链霉菌原核生物，是伽马类的原型，也是第一个从厌氧生物或古生界被鉴定的碳酸酐酶。这四个类别的所有酶都经过了好氧纯化，据报道含有催化锌。在这里，我们报道了apo-CaM与Fe2+的厌氧重组，得到了有效k(CAT)超过锌重组酶的CaM。穆斯堡尔谱显示，Fe2+重组酶含有高自旋的Fe2+，当氧化为Fe3+时，该酶失活。与Fe3+的重组不成功。与Cu2+、Mn2+、Ni2+或Cd2+重组产生的酶的有效k(CAT)值为10%或小于锌离子重组CaM的值。在大肠杆菌中生产的CaM和纯化的厌氧含铁的CaM的有效k(CAT)和k(CAT)/K-m值超过了锌重组CaM的值。研究结果证实了铁的一种以前不为人知的生物学功能。
Since 1933, carbonic anhydrase research has focused on enzymes from mammals (alpha class) and plants (beta class); however, two additional classes (gamma and delta) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the gamma class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. Herein, we report the anaerobic reconstitution of apo-Cam with Fe2+, which yielded Cam with an effective k(cat) that exceeded that for the Zn2+-reconstituted enzyme. Mossbauer spectroscopy showed that the Fe2+-reconstituted enzyme contained high spin Fe2+ that, when oxidized to Fe3+, inactivated the enzyme. Reconstitution with Fe3+ was unsuccessful. Reconstitution with Cu2+, Mn2+, Ni2+, or Cd2+ yielded enzymes with effective k(cat), values that were 10% or less than the value for the Zn2+-reconstituted Cam. Cam produced in Escherichia coli and purified anaerobically contained iron with effective k(cat) and k(cat)/K-m values exceeding the values for Zn2+-reconstituted Cam. The results identify a previously unrecognized biological function for iron.