Ferrochelatase π-helix: Implications from examining the role of the conserved π-helix glutamates in porphyrin metalation and product release.
Ferrochelatase π-helix: Implications from examining the role of the conserved π-helix glutamates in porphyrin metalation and product release.
复制标题
铁螯合酶α-螺旋：检查保守的α-螺旋谷氨酸在卟啉金属化和产品释放中的作用的意义。
DOI：
10.1016/j.abb.2018.02.015
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发表时间：
2018
影响因子：
3.9
通讯作者：
Ferreira,GloriaC
中科院分区：
文献类型：
--
作者：
Gillam,MalloryE;Hunter,GregoryA;Ferreira,GloriaC
原卟啉铁螯合酶催化Fe2+插入原卟啉IX形成血红素。为了确定一个保守的活性位点π-螺旋是否有助于金属离子底物向铁螯合酶结合的卟啉底物的移位，将不变的π-螺旋谷氨酸替换为具有非带负电荷侧链的氨基酸，并对产生的变异的动力学机制进行了研究。对酵母野生型铁螯合酶-、E314Q-和e318q -在多次和单次转化条件下催化的反应的分析表明，π-螺旋谷氨酸的突变阻碍了原卟啉金属化和金属化卟啉的释放，每一步都减慢了大约30-50%。原卟啉金属化的表观pkaf为7.3 ± 0.1，这是由去质子化的Glu-314和Glu-314辅助的Fe2+插入到卟啉环中导致的。我们提出，π螺旋的解绕伴随着蛋白质开放构象的采用，使去质子化的glue -314从酶的表面结合Fe2+。过渡到封闭构象，带π螺旋缠绕，将glu -314结合的Fe2+带到活性位点并入原卟啉。
Protoporphyrin ferrochelatase catalyzes the insertion of Fe2+into protoporphyrin IX to form heme. To determine whether a conserved, active site π-helix contributes to the translocation of the metal ion substrate to the ferrochelatase-bound porphyrin substrate, the invariant π-helix glutamates were replaced with amino acids with non-negatively charged side chains, and the kinetic mechanisms of the generated variants were examined. Analysis of yeast wild-type ferrochelatase-, E314Q- and E318Q-catalyzed reactions, under multi- and single-turnover conditions, demonstrated that the mutations of the π-helix glutamates hindered both protoporphyrin metalation and release of the metalated porphyrin, by slowing each step by approximately 30–50%. Protoporphyrin metalation occurred with an apparent pKaof 7.3 ± 0.1, which was assigned to binding of Fe2+by deprotonated Glu-314 and Glu-314-assisted Fe2+insertion into the porphyrin ring. We propose that unwinding of the π-helix concomitant with the adoption of a protein open conformation positions the deprotonated Glu-314 to bind Fe2+from the surface of the enzyme. Transition to the closed conformation, with π-helix winding, brings Glu-314-bound Fe2+to the active site for incorporation into protoporphyrin.