Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase.
Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase.
复制标题
牛肝尿卟啉原脱羧酶的纯化和表征。
DOI：
10.1021/bi00289a009
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发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
Kushner,JP
中科院分区：
文献类型：
--
作者：
Straka,JG;Kushner,JP
James G. Straka* 和James P. Kushner* 摘要：尿卟啉原脱羧酶（EC 4.1. 1.37）已经通过使用等电和盐沉淀，然后通过在DEAE-纤维素、苯基-琼脂糖、羟基磷灰石和Sephacryl S-200上的色谱法从牛肝中纯化至均一。纯化的酶是单体，具有~ 57 000和在pH 4.6的等电点。酶活性在离子强度约为0.1 M且pH为6.8的缓冲液中最佳。纯化的酶具有936 nmol·h-1·（mg蛋白质）-1的比活性（表示为尿卟啉原I的消失）。纯化的酶催化尿卟啉原I或III转化为相应粪卟啉原的所有四个脱羧反应。尿卟啉原III向粪卟啉原III的生理学显着转化中的限速步骤是七羧酸盐III的脱羧。动力学数据表明，该酶至少有两个非相互作用的活性位点。催化活性需要至少一个巯基。该酶被巯基特异性试剂和二价金属离子抑制，并通过胞质酶尿卟啉原脱羧酶（EC 4.1）将尿卟啉原（八羧酸卟啉原）转化为粪卟啉原（四羧酸卟啉原）。1.37）（伯纳姆，1969年）。尿卟啉原的四条醋酸酯侧链从“D”环开始顺时针顺序脱羧。生成具有七个、六个和五个羧酸酯取代基的中间体化合物（杰克逊等人，1976年）。虽然尿卟啉原III在生理条件下是脱羧酶的底物，但尿卟啉原的两种天然存在的异构体（I和III）都可以作为底物。因此，该酶共有八种潜在底物和八种反应产物：
James G. Straka* and James P. Kushner* abstract: Uroporphyrinogen decarboxylase (EC 4.1. 1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200. The purified enzyme is a monomer with an~ 57 000 and an isoelectric point at pH 4.6. Enzyme activity is optimal in buffers having an ionic strength of~ 0.1 M and a pH of 6.8. The purified enzyme has a specific activity (expressed as the disappearance of uroporphyrinogen I) of 936 nmol-h-1-(mg of protein)-1. The purified enzyme catalyzes all four decarboxylation reactions in the conversion of uro-porphyrinogen I or III to the corresponding coproporphyrinogen. The rate-limiting step in the physiologically significant conversion of uroporphyrinogen III to coproporphyrinogen III is the decarboxylation of heptacarboxylate III. Kinetic data suggest that the enzyme has at least two noninteracting active sites. At least one sulfhydryl group is required for catalytic activity. The enzyme is inhibited by sulfhydryl-specific reagents and by divalent metal ions inroporphyrinogen (an octacarboxylate porphyrinogen) is converted to coproporphyrinogen (a tetracarboxylate porphyrinogen) by the cytosolic enzyme uroporphyrinogen decarboxylase (EC 4.1. 1.37)(Burnham, 1969). The four acetate side chains of uroporphyrinogen are decarboxylated sequen-tially in a clockwise fashion starting on the “D” ring. Inter-mediate compounds with seven, six, and five carboxylate substituents are generated (Jackson et al., 1976). Although uroporphyrinogen III is the substrate for decarboxylase under physiologic conditions, both naturally occurring isomers of uroporphyrinogen (I and III) may serve as substrates. Thus, there are a total of eight potential substrates and eight reaction products for the enzyme: