SIGNIFICANT CONTRIBUTION OF ARGININE-112 AND ITS POSITIVE CHARGE OF PSEUDOMONAS-PUTIDA CYTOCHROME P-450(CAM) IN THE ELECTRON-TRANSPORT FROM PUTIDAREDOXIN
SIGNIFICANT CONTRIBUTION OF ARGININE-112 AND ITS POSITIVE CHARGE OF PSEUDOMONAS-PUTIDA CYTOCHROME P-450(CAM) IN THE ELECTRON-TRANSPORT FROM PUTIDAREDOXIN
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DOI：
10.1016/0167-4838(94)90049-3
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发表时间：
1994-07-20
期刊：
影响因子：
--
通讯作者：
SAGARA, Y
中科院分区：
文献类型：
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作者：
NAKAMURA, K;HORIUCHI, T;SAGARA, Y
恶臭假单胞菌的细胞色素P-450（cam）是多种真核细胞色素P-450分子的原型。位于该蛋白表面的Arg-112在各种其他细胞色素P-450中高度保守。在这项研究中，我们构建了突变基因的P-450（cam），其中Arg-112被替换为谷氨酰胺或谷氨酸，他们在大肠杆菌中表达和纯化的突变蛋白。在重组系统中分析它们的P-450酶活性以确定Arg-112的功能。Arg（112）-Gln和Arg（112）-Glu的d-樟脑的Kd值与野生型酶的Kd值大致相同，而putidaredoxin的氧化形式的Kd值分别为240和530 μ M，putidaredoxin是一种酸性蛋白质，是P-450（cam）的氧化还原配偶体。这些值比野生型酶（28 μ M）大8和19倍，从而表明突变酶对氧化的putidaredoxin的亲和力较低。采用停流法测定的还原型putidaredoxin还原的反应速率常数，野生型、Arg（112）-Gln和Arg（112）-Glu分别为45.5、9.0.10（-3）和9.0.10（-4）s（-1）。因此，P-450（cam）的Arg-112在与putidaredoxin的相互作用和高效率的电子转移中起着重要作用;残基的正电荷似乎有助于该过程。在大肠杆菌中的产量、纯化组分中血红素含量和突变蛋白的热稳定性均低于野生型酶，表明P-450（cam）的Arg-112对P-450（cam）的稳定性也很重要。
Cytochrome P-450(cam) of Pseudomonas putida is a prototype of various eukaryotic cytochrome P-450 molecules. Arg-112 located on the surface of this protein is highly conserved among various other cytochromes P-450. In this study, we constructed mutant genes for P-450(cam) in which Arg-112 was replaced by Gln or Glu, expressed them in Escherichia coli and purified the mutant proteins. Their P-450 enzymic activities were analyzed in the reconstituted system to determine the function of Arg-112. K-d values for d-camphor of Arg(112)-Gln and Arg(112)-Glu were much the same as those of the wild-type enzyme, whereas K-d values for the oxidized form of putidaredoxin, which is an acidic protein and is the redox partner of P-450(cam) were 240 and 530 mu M, respectively. These values are 8 and 19 times larger than that of the wild-type enzyme (28 mu M), thereby indicating lower affinities of the mutant enzymes for the oxidized putidaredoxin. Reaction rate constants for reduction by the reduced form of putidaredoxin, measured using the stopped flow method, were 45.5, 9.0.10(-3) and 9.0.10(-4) s(-1) for the wild type, Arg(112)-Gln and Arg(112)-Glu, respectively. Thus, Arg-112 of P-450(cam) plays an important role in the interaction with putidaredoxin and in the high efficiency of the electron transfer; the positive charge of the residue seeming to contribute to the process. The yields in Escherichia coli, the heme contents in the purified fractions and heat stability of the mutant proteins were lower than those of the wild type enzyme, suggesting that Arg-112 of P-450(cam) is also important for stability of P-450(cam).