Samson S M, Chapman J L, Belagaje R, Queener S W, Ingolia T D
Proc Natl Acad Sci U S A. 1987 Aug;84(16):5705-9. doi: 10.1073/pnas.84.16.5705.
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.
顶头孢霉和产黄青霉异青霉素N合成酶的预测氨基酸序列在类似位置有两个半胱氨酸残基（按顶头孢霉编号为Cys-106和Cys-255）。为了研究这些半胱氨酸残基在顶头孢霉该酶活性中的作用，我们使用定点体外诱变将这些半胱氨酸残基变为丝氨酸残基。Cys-255的突变使比活性降低约50%，而Cys-106的突变或Cys-106和Cys-255两者的突变使比活性降低约97%。这表明半胱氨酸对于异青霉素N合成酶活性很重要但并非必不可少。异青霉素N合成酶的烷基化也几乎完全使该酶失活，但残余活性可能是由于烷基化不完全所致。在这种情况下，通过基因操作进行原子取代是评估巯基部分在酶活性中作用的更准确方法。
