Bovine ferrochelatase. Kinetic analysis of inhibition by N-methylprotoporphyrin, manganese, and heme.
Bovine ferrochelatase. Kinetic analysis of inhibition by N-methylprotoporphyrin, manganese, and heme.
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DOI：
10.1016/s0021-9258(17)44247-5
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发表时间：
1983-10
期刊：
影响因子：
--
通讯作者：
H. Dailey;J. Fleming
中科院分区：
文献类型：
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作者：
H. Dailey;J. Fleming
血红素生物合成途径的末端酶亚铁螯合酶（原血红素亚铁裂解酶EC 4.99.1.1）已经使用与Taketani和Tokunaga（Taketani，S.和Tokunaga，R.（1981）J.Biol.Chem.256，12748-12753）用于从大鼠肝脏纯化酶。纯化倍数为2000倍时，最终收率为49%。在0.5%胆酸钠存在下，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Sepharose CL-6 B柱层析，铁螯合酶的表观分子量约为40，000。纯化的酶只有轻微的刺激，添加脂质和抑制Mn ~（2+），Pb ~（2+），Hg ~（2+）。牛亚铁螯合酶利用原卟啉、中卟啉和次卟啉，但不利用二取代卟啉（2，4-二磺酸和2，4-二甘醇次卟啉）。N-甲基原卟啉，一些药物代谢的有毒副产物，被发现以竞争性方式抑制亚铁螯合酶，相对于卟啉，Ki为7 nM，相对于铁无竞争性。锰抑制亚铁螯合酶竞争性相对于铁（Ki = 15 μ M）和非竞争性相对于卟啉底物。血红素是铁的非竞争性抑制剂。这些研究结果导致一个连续的双双动力学模型与铁螯合酶与铁结合发生之前，卟啉结合和血红素被释放之前，释放两个质子。
The terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase EC 4.99.1.1), has been purified to apparent homogeneity from bovine liver mitochondria using a scheme similar to that reported by Taketani and Tokunaga (Taketani, S. and Tokunaga, R. (1981) J. Biol. Chem. 256, 12748-12753) for purification of the enzyme from rat liver. The final yield was 49% with a 2000-fold purification. Ferrochelatase has an apparent molecular weight of approximately 40,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and column chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. The purified enzyme was only slightly stimulated by added lipid and was inhibited by Mn2+, Pb2+, and Hg2+. Bovine ferrochelatase utilized proto-, meso-, and deuteroporphyrin, but not disubstituted porphyrins (2,4-disulfonic and 2,4-bisglycol deuteroporphyrin). N-Methylprotoporphyrin, a toxic by-product of the metabolism of some drugs, was found to inhibit ferrochelatase in a competitive fashion with respect to porphyrin with a Ki of 7 nM and uncompetitive with respect to iron. Manganese inhibits ferrochelatase competitively with respect to iron (Ki = 15 microM) and noncompetitively with respect to the porphyrin substrate. Heme, one of the products, is a noncompetitive inhibitor with respect to iron. These findings lead to a sequential Bi Bi kinetic model for ferrochelatase with iron binding occurring prior to porphyrin binding and heme being released prior to the release of two protons.