Blom C, Klasen E C, Van Steveninck J
Sylvius Laboratories, Department of Medical Biochemistry, Leiden, The Netherlands.
Biochim Biophys Acta. 1990 Jul 6;1039(3):339-42. doi: 10.1016/0167-4838(90)90268-k.
Ferrochelatase activity was measured in crude extracts of fibroblasts, obtained from erythropoietic protoporphyria patients and healthy controls. The enzyme activity in erythropoietic protoporphyria fibroblasts was about 50% lower, compared to the controls. The sulfhydryl-oxidising reagent diamide inhibited the normal enzyme by about 50%, whereas ferrochelatase from erythropoietic protoporphyria fibroblasts was completely insensitive to the reagent. Pb2+ inhibits ferrochelatase activity by reacting with essential sulfhydryl groups. Low concentrations of Pb2+ inhibited the normal enzyme by 56%, but the mutant enzyme by only 8%. The photodynamic activity of bound mesoporphyrin substrate caused a biphasic inactivation of the normal enzyme. During the first 5 min of illumination a fast decrease of enzyme activity occurred to about 60% of the initial value. Experimental evidence indicates that this first phase of inactivation is caused by photooxidation of sulfhydryl groups. During further illumination inactivation continued at a much slower rate. With ferrochelatase from erythropoietic protoporphyria fibroblasts only the second, slow phase of photodynamic inactivation was observed. These observations suggest a mutation of ferrochelatase in erythropoietic protoporphyria, affecting the reactivity of sulfhydryl groups, involved in the catalytic activity of the enzyme.
测定了从红细胞生成性原卟啉症患者和健康对照者获取的成纤维细胞粗提物中的亚铁螯合酶活性。与对照组相比，红细胞生成性原卟啉症成纤维细胞中的酶活性降低了约50%。巯基氧化试剂二酰胺对正常酶的抑制作用约为50%，而红细胞生成性原卟啉症成纤维细胞中的亚铁螯合酶对该试剂完全不敏感。Pb2+通过与必需的巯基反应来抑制亚铁螯合酶活性。低浓度的Pb2+对正常酶的抑制率为56%，而对突变酶的抑制率仅为8%。结合的中卟啉底物的光动力活性导致正常酶出现双相失活。在光照的最初5分钟内，酶活性迅速下降至初始值的约60%。实验证据表明，失活的第一阶段是由巯基的光氧化引起的。在进一步光照期间，失活以慢得多的速率继续。对于红细胞生成性原卟啉症成纤维细胞中的亚铁螯合酶，仅观察到光动力失活的第二个缓慢阶段。这些观察结果表明红细胞生成性原卟啉症中亚铁螯合酶发生了突变，影响了参与酶催化活性的巯基的反应性。
