Shi Zhen, Ferreira Gloria C
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA.
Biochem J. 2006 Oct 1;399(1):21-8. doi: 10.1042/BJ20060753.
Protoporhyrin IX ferrochelatase catalyses the terminal step of the haem-biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. NMPP (N-methylprotoporphyrin), a transition-state analogue and potent inhibitor of ferrochelatase, is commonly used to induce haem deficiency in mammalian cell cultures. To create ferrochelatase variants with different extents of tolerance towards NMPP and to understand further the mechanism of ferrochelatase inhibition by NMPP, we isolated variants with increased NMPP resistance, bearing mutations in an active-site loop (murine ferrochelatase residues 248-257), which was previously shown to mediate a protein conformational change triggered by porphyrin binding. The kinetic mechanisms of inhibition of two variants, in which Pro255 was replaced with either arginine (P255R) or glycine (P255G), were investigated and compared with that of wild-type ferrochelatase. While the binding affinity of the P255X variants for NMPP decreased by one order of magnitude in relation to that of wild-type enzyme, the inhibition constant increased by approximately two orders of magnitude (K(i)(app) values of 1 microM and 2.3 microM for P255R and P255G respectively, as against 3 nM for wild-type ferrochelatase). Nonetheless, the drastically reduced inhibition of the variants by NMPP was not paralleled with a decrease in specificity constant (kcat/K(m, protoporhyrin IX)) and/or catalytic activity (kcat). Further, although NMPP binding to either wild-type ferrochelatase or P255R occurred via a similar two-step kinetic mechanism, the forward and reverse rate constants associated with the second and rate-limiting step were comparable for the two enzymes. Collectively, these results suggest that Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase.
原卟啉 IX 亚铁螯合酶通过将亚铁插入原卟啉 IX 来催化血红素生物合成途径的最后一步。NMPP（N-甲基原卟啉）是一种过渡态类似物，也是亚铁螯合酶的有效抑制剂，常用于在哺乳动物细胞培养物中诱导血红素缺乏。为了创建对 NMPP 具有不同耐受程度的亚铁螯合酶变体，并进一步了解 NMPP 抑制亚铁螯合酶的机制，我们分离出了对 NMPP 具有更高抗性的变体，这些变体在一个活性位点环（小鼠亚铁螯合酶残基 248 - 257）中发生了突变，先前已表明该环介导由卟啉结合引发的蛋白质构象变化。研究了两个变体（其中 Pro255 分别被精氨酸（P255R）或甘氨酸（P255G）取代）的抑制动力学机制，并与野生型亚铁螯合酶进行了比较。虽然 P255X 变体对 NMPP 的结合亲和力相对于野生型酶降低了一个数量级，但抑制常数增加了约两个数量级（P255R 和 P255G 的 K(i)(app)值分别为 1 μM 和 2.3 μM，而野生型亚铁螯合酶为 3 nM）。尽管如此，NMPP 对变体的抑制作用大幅降低，但特异性常数（kcat/K(m,原卟啉 IX)）和/或催化活性（kcat）并未随之降低。此外，虽然 NMPP 与野生型亚铁螯合酶或 P255R 的结合都通过类似的两步动力学机制发生，但与第二步和限速步骤相关的正向和反向速率常数在两种酶中相当。总体而言，这些结果表明 Pro255 在维持适当的蛋白质构象以及调节亚铁螯合酶的选择性和/或区域特异性方面起着关键作用。
