Davidson Ruth E, Chesters Christopher J, Reid James D
Department of Chemistry, University of Sheffield, Sheffield S3 7HF, United Kingdom.
J Biol Chem. 2009 Dec 4;284(49):33795-9. doi: 10.1074/jbc.M109.030205. Epub 2009 Sep 19.
Protoporphyrin IX ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Ferrochelatase shows specificity, in vitro, for multiple metal ion substrates and exhibits substrate inhibition in the case of zinc, copper, cobalt, and nickel. Zinc is the most biologically significant of these; when iron is depleted, zinc porphyrins are formed physiologically. Examining the k(cat)/K(m)(app) ratios for zinc and iron reveals that, in vitro, zinc is the preferred substrate at all concentrations of porphyrin. This is not the observed biological specificity, where zinc porphyrins are abnormal; these data argue for the existence of a specific iron delivery mechanism in vivo. We demonstrate that zinc acts as an uncompetitive substrate inhibitor, suggesting that ferrochelatase acts via an ordered pathway. Steady-state characterization demonstrates that the apparent k(cat) depends on zinc and shows substrate inhibition. Although porphyrin substrate is not inhibitory, zinc inhibition is enhanced by increasing porphyrin concentration. This indicates that zinc inhibits by binding to an enzyme-product complex (EZnD(IX)) and is likely to be the second substrate in an ordered mechanism. Our analysis shows that substrate inhibition by zinc is not a mechanism that can promote specificity for iron over zinc, but is instead one that will reduce the production of all metalloporphyrins in the presence of high concentrations of zinc.
原卟啉IX亚铁螯合酶（EC 4.99.1.1）催化血红素生物合成途径的最后一步，即将亚铁插入原卟啉IX中。亚铁螯合酶在体外对多种金属离子底物具有特异性，并且在锌、铜、钴和镍存在的情况下表现出底物抑制作用。锌是这些金属离子中生物学意义最为显著的；当铁缺乏时，生理上会形成锌卟啉。检查锌和铁的k(cat)/K(m)(app)比值发现，在体外，在所有卟啉浓度下锌都是首选底物。这与观察到的生物学特异性不同，在生物学中锌卟啉是异常的；这些数据表明体内存在一种特定的铁传递机制。我们证明锌作为非竞争性底物抑制剂，这表明亚铁螯合酶通过有序途径起作用。稳态特征表明表观k(cat)取决于锌并表现出底物抑制作用。尽管卟啉底物没有抑制作用，但增加卟啉浓度会增强锌的抑制作用。这表明锌通过与酶-产物复合物（EZnD(IX)）结合而产生抑制作用，并且可能是有序机制中的第二种底物。我们的分析表明，锌的底物抑制作用不是一种能够促进对铁而非锌具有特异性的机制，而是一种在高浓度锌存在下会降低所有金属卟啉生成的机制。
