Substitutions in the C-terminal portion of the catalytic domain partially reverse assembly defects introduced by mutations in the N-terminal linker sequence of cytochrome P450 2C2.
Substitutions in the C-terminal portion of the catalytic domain partially reverse assembly defects introduced by mutations in the N-terminal linker sequence of cytochrome P450 2C2.
复制标题
催化结构域 C 端部分的取代部分逆转了由细胞色素 P450 2C2 N 端接头序列突变引起的组装缺陷。
DOI：
10.1021/bi9906266
复制
发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Kemper,B
中科院分区：
文献类型：
--
作者：
Doray,B;Chen,CD;Kemper,B
细胞色素P450 2C 2的N-末端信号锚序列之后的7-氨基酸连接片段中的突变已显示影响血红素蛋白的正确组装并降低COS细胞中表达的突变体的活性。相反，C2 pmBalC 1，其中细胞色素P450 2C 1残基取代的细胞色素P450 2C 2的C-末端区域，表现出增加的活性时，在COS-1细胞中表达。为了进一步研究C2 pmBalC 1在COS-1细胞中活性增加的基础，在昆虫细胞和大肠杆菌中表达了该蛋白。在这些系统中表达的C2 pmBalC 1的功能性P450种类的量和P450与P420的比率大于细胞色素P450 2C 2的量，表明更有效的组装是C2 pmBalC 1活性增加的基础。为了确定C-末端取代是否可以补偿由N-末端接头突变介导的组装减少，将接头突变引入C2 pmBalCl中。如果接头中的所有7个氨基酸缺失，则在表达突变体的COS-1、昆虫或细菌细胞中未检测到酶活性细胞色素P450 2C 2或C2 pmBalC 1。突变体C2 A2，其中两个丙氨酸取代的接头，在COS-1细胞中没有检测到的月桂酸羟化酶活性，和少量的血红素蛋白，为这个突变体表达在E。大肠杆菌和昆虫细胞。相反，C2 pmBalC 1中的相同突变在COS-1细胞中仅降低了50%的活性，并且在细菌和昆虫细胞中显著提高了P450表达水平。A2突变不影响在昆虫细胞的全细胞裂解物中测定的细胞色素P450 2C 2或C2 pmBalC 1的酶活性，但降低了在重构测定系统中测定的部分纯化的酶的活性。这些发现表明，引入到P450 2C 2的C-末端区域的突变可以促进蛋白质的组装，并部分逆转由N-末端突变导致的组装减少。
Mutations in a 7-amino acid linker segment, immediately following the N-terminal signal anchor sequence of cytochrome P450 2C2, have been shown to affect proper assembly of hemoprotein and decrease activity of the mutants expressed in COS cells. In contrast, C2pmBalC1, in which cytochrome P450 2C1 residues were substituted for those of cytochrome P450 2C2 in the C-terminal region, exhibited increased activity when expressed in COS-1 cells. To examine further the basis for the increased activity of C2pmBalC1 in COS-1 cells, the protein was expressed in insect cells andEscherichia coli. The amounts of the functional P450 species of C2pmBalC1 expressed in these systems and the ratios of P450 to P420 were greater than those of cytochrome P450 2C2, indicating that more efficient assembly underlies the increased activity of C2pmBalC1. To determine whether the C-terminal substitutions could compensate for the decreased assembly mediated by the N-terminal linker mutations, the linker mutations were introduced into C2pmBalC1. If all 7 amino acids in the linker were deleted, no enzymatically active cytochrome P450 2C2 or C2pmBalC1 was detected in COS-1, insect, or bacterial cells expressing the mutants. The mutant C2A2, in which two alanines were substituted for the linker, had no detectable laurate hydroxylase activity in COS-1 cells, and minor amounts of hemoprotein for this mutant were expressed inE. coliand insect cells. In contrast, the same mutation in C2pmBalC1 reduced activity only 50% in COS-1 cells and markedly elevated levels of P450 expression in bacteria and insect cells. The A2 mutation did not affect the enzymatic activity of either cytochrome P450 2C2 or C2pmBalC1 assayed in whole cell lysates of insect cells but reduced the activity of partially purified enzymes assayed in a reconstituted assay system. These findings indicate that mutations introduced into the C-terminal region of P450 2C2 can facilitate assembly of the proteins and partially reverse the decreased assembly resulting from the N-terminal mutations.