Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues.
Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues.
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DOI：
10.1021/bi010012c
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发表时间：
2001-07
期刊：
影响因子：
2.9
通讯作者：
V. M. Sellers;Chia-Kuei Wu;T. Dailey;H. Dailey
中科院分区：
文献类型：
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作者：
V. M. Sellers;Chia-Kuei Wu;T. Dailey;H. Dailey
血红素生物合成的最后一步，即亚铁插入原卟啉IX形成原血红素，是由铁络合酶(EC 4.99.1.1)催化的。通过定点突变，从人铁络合酶的晶体结构中鉴定出一些高度保守的残基，它们位于活性部位。突变株Y123F、Y165F、Y191H和R164L对铁的K(M)增加，对卟啉的K(M)没有改变。双突变体R164L/Y165F对铁的K(M)增加了6倍，V(Max)降低了10倍。双突变体Y123F/Y191F活性较低，K(M)升高，Y123F/Y165F没有可测到的活性。突变体H263A/C/N、D340N、E343Q、E343H和E343K没有检测到酶活性，而E343D、E347Q和H341C降低了S的V(Max)，而两种底物的K(M)S都没有显著变化。D340E具有接近正常的动力学参数，而D383A和H231A增加了铁的K(M)S。根据这些数据和人的铁络合酶的晶体结构，提出了E343、H341和D340残基构成了从活性部位的H263到蛋白质表面的通道，并在从卟啉大环中提取质子的过程中发挥了作用。由于金属化作用只发生在质子提取的过程中，因此作为卟啉质子接受残基的H263的作用是催化的中心。这表明，铁是通过W227和Y191残基从D383/H231的酶外部转运到活性中心口袋相反的R164和Y165残基的金属化位置。这个模型对于线粒体膜相关的真核细胞铁络合酶应该是通用的，但对于细菌铁络合酶可能不同，因为酶在原核细胞中的空间定向可能不同。
The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.