Kinetics and inhibition of recombinant human cystathionine γ-lyase -: Toward the rational control of transsulfuration
Kinetics and inhibition of recombinant human cystathionine γ-lyase -: Toward the rational control of transsulfuration
复制标题
DOI：
10.1074/jbc.274.18.12675
复制
发表时间：
1999-04-30
影响因子：
4.8
通讯作者：
Wahl, MC
中科院分区：
文献类型：
--
作者：
Steegborn, C;Clausen, T;Wahl, MC
从细胞内Rep G2总RNA中克隆了人胱硫醚-伽马裂解酶基因。融合到T7启动子允许在大肠杆菌中表达，这是第一个在细菌系统中过量生产的哺乳动物半胱氨酸伽马裂解酶。约90%的异源基因产物是不溶的，从纯化的包涵体中复性实验的成功率有限。通过三步自然过程，也可以从可溶性裂解组分中提取约5 mg/升培养的人胱硫氨酸伽马裂解酶。该酶对L-半胱氨酸(K-m=0.5 mM，V-max=2.5U/mg)有较高的伽马裂解酶活力，最适pH为8.2，对L-半胱氨酸和L-半胱氨酸没有残留的β-裂解酶行为，仅有边缘反应。用基于机理的灭活剂丙炔甘氨酸、三氟丙氨酸和氨基乙氧基乙烯基甘氨酸进行了抑制研究。丙叉甘氨酸对人胱硫氨酸伽玛裂解酶的失活比三氟丙氨酸要强得多，这与该酶对C-伽马-S键的偏好一致。氨基乙氧基乙烯基甘氨酸表现出缓慢而紧密的结合特征，其K-I为10.5mU M，与其对胱硫醚β-裂解酶的作用相当，这一结果对设计硫化组分的特异性抑制剂具有重要意义。
The gene encoding human cystathionine gamma-lyase was cloned from total cellular Rep G2 RNA. Fusion to a T7 promoter allowed expression in Escherichia coli, representing the first mammalian cystathionine gamma-lyase overproduced in a bacterial system. About 90% of the heterologous gene product was insoluble, and renaturation experiments from purified inclusion bodies met with limited success. About 5 mg/liter culture of human cystathionine gamma-lyase could also be extracted from the soluble lysis fraction, employing a three-step native procedure. While the enzyme showed high gamma-lyase activity toward L-cystathionine (K-m = 0.5 mM, V-max = 2.5 units/mg) with an optimum pH of 8.2, no residual cystathionine beta-lyase behavior and only marginal reactivity toward L-cystine and L-cysteine were detected. Inhibition studies were performed with the mechanism-based inactivators propargylglycine, trifluoroalanine, and aminoethoxyvinylglycine. Propargylglycine inactivated human cystathionine gamma-lyase much more strongly than trifluoroalanine, in agreement with the enzyme's preference for C-gamma-S bonds. Aminoethoxyvinylglycine showed slow and tight binding characteristics with a K-i of 10.5 mu M, comparable with its effect on cystathionine beta-lyase, The results have important implications for the design of specific inhibitors for transsulfuration components.