Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis.
Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis.
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通过定点诱变分析半胱氨酸残基在异青霉素 N 合成酶活性中的作用。
DOI：
10.1073/pnas.84.16.5705
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发表时间：
1987
影响因子：
11.1
通讯作者：
T. D. Ingolia
中科院分区：
文献类型：
--
作者：
S. Samson;J. Chapman;R. Belagaje;S. Queener;T. D. Ingolia
顶顶头孢霉和产黄青霉的异青霉素N合成酶的预测氨基酸序列在相似的位置都有两个半胱氨酸残基(顶顶头孢霉编号为Cys-106和Cys-255)。为了研究这些半胱氨酸残基在顶顶藻酶活力中的作用，我们使用了体外定点突变的方法将这些半胱氨酸残基转化为丝氨酸残基。Cys-255的突变使比活性降低约50%，而Cys-106的突变或Cys-106和Cys-255的突变使比活性降低约97%。这表明半胱氨酸对IPNS的活性是重要的，但不是必需的。IPNS的烷基化也几乎完全灭活了该酶，但残留的活性可能是由于烷基化不完全所致。在这种情况下，通过遗传操作进行原子取代是一种更准确地评估巯基部分在酶活性中的作用的方法。
The predicted amino acid sequences of isopenicillin N synthetase from both Cephalosporium acremonium and Penicillium chrysogenum have two cysteine residues in analogous positions (Cys-106 and Cys-255 in the C. acremonium numbering). To examine the role of these cysteine residues in the activity of the C. acremonium enzyme, we used site-directed in vitro mutagenesis to change these cysteine residues to serine residues. Mutation of Cys-255 reduces specific activity approximately equal to 50%, whereas mutation of Cys-106 or mutation of both Cys-106 and Cys-255 reduces specific activity about 97%. This suggests that the cysteines are important but not essential for IPNS activity. Alkylation of IPNS also almost completely inactivated the enzyme, but residual activity could have been due to incomplete alkylation. Atomic substitution via genetic manipulation in this case is a more accurate means of assessing the role of sulfhydryl moieties in enzyme activity.