CHANGING THE HYDROGEN-BONDING POTENTIAL IN THE DNA-BINDING SITE OF ECORI BY SITE-DIRECTED MUTAGENESIS DRASTICALLY REDUCES THE ENZYMATIC-ACTIVITY, NOT, HOWEVER, THE PREFERENCE OF THIS RESTRICTION ENDONUCLEASE FOR CLEAVAGE WITHIN THE SITE -GAATTC-
CHANGING THE HYDROGEN-BONDING POTENTIAL IN THE DNA-BINDING SITE OF ECORI BY SITE-DIRECTED MUTAGENESIS DRASTICALLY REDUCES THE ENZYMATIC-ACTIVITY, NOT, HOWEVER, THE PREFERENCE OF THIS RESTRICTION ENDONUCLEASE FOR CLEAVAGE WITHIN THE SITE -GAATTC-
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DOI：
10.1021/bi00432a047
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发表时间：
1989-03-21
期刊：
影响因子：
2.9
通讯作者：
PINGOUD, A
中科院分区：
文献类型：
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作者：
ALVES, J;RUTER, T;PINGOUD, A
根据EcoRI-寡脱氧核苷酸复合物的X射线结构分析[McClarin等（1986）Science 234，1526]，序列特异性由12个氢键介导，从二聚体酶的两个相同亚基中的每一个到识别位点-GAATTC-有6个氢键：Arg 200与鸟嘌呤形成两个氢键，而Glu 144和Arg 145与相邻的腺嘌呤残基形成四个氢键。改变识别位点处的氢键势而不干扰界面的其余部分应导致简并序列的识别[Rosenberg等人（1987）in Protein Engineering（Oxender，D. L.，和Fox，C. F.、编辑）第237-250页，利斯，纽约]。我们以前已经证明，用Gln代替Glu 144和用Lys代替Arg 145会影响酶的活性，但不会影响其特异性[Wolfes等（1986）Nucleic Acids Res.14，9063]。我们现在表明，Arg 200突变为Lys，Glu 144 Arg 145突变为GlnLys，以及Glu 144 Arg 145 Arg 200突变为GlnLysLys的三重突变也不会导致EcoRI切割特异性的可检测退化，但会显著损害该酶的催化活性。裂解的pUC 8 DNA和一个tridecadeoxynucleotide底物的稳态动力学的详细分析表明，到目前为止研究的所有DNA结合位点突变体的活性降低主要是由于kcat的减少，除了Arg 200到Lys突变体，这是只在其KM受损。硝酸纤维素滤膜实验证实了这一观察结果，该实验表明，该突变体对寡脱氧核苷酸底物的亲和力比其它DNA结合位点突变体低，而亲和力比野生型EcoRI低得多。虽然这些和以前的数据清楚地表明，Glu 144，Arg 145和Arg 200是必要的有效的底物结合和催化，它们参与的具体识别是更复杂的比提出的基础上的X-射线结构分析。我们的研究结果还表明，EcoRI的特异性不能轻易放松，特别是不影响其活性。
According to the X-ray structure analysis of an EcoRI-oligodeoxynucleotide complex [McClarin et al. (1986) Science 234, 1526], sequence specificity is mediated by 12 hydrogen bonds, 6 from each of the two identical subunits of the dimeric enzyme to the recognition site-GAATTC-: Arg200 forms two hydrogen bonds with guanine, while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. Changing the hydrogen-bonding potential at the recognition site without perturbing the rest of the interface should lead to the recognition of degenerate sequences [Rosenberg et al. (1987) in Protein Engineering (Oxender, D. L., and Fox, C. F., Eds.) pp 237-250, Liss, New York]. We have shown previously that replacing Glu144 by Gln and Arg145 by Lys affects the activity of the enzyme, not, however, its specificity [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. We show now that also the mutation of Arg200 to Lys, the double mutation Glu144Arg145 to GlnLys, and the triple mutation Glu144Arg145Arg200 to GlnLysLys do not lead to a detectable degeneracy of the specificity of cleavage by EcoRI but significantly impair the catalytic activity of this enzyme. A detailed analysis of the steady-state kinetics of cleavage of pUC8 DNA and a tridecadeoxynucleotide substrate demonstrates that the reduction in activity for all DNA binding site mutants investigated so far is mainly due to a decrease in kcat, with the exception of the Arg200 to Lys mutant, which is only impaired in its KM. This observation is confirmed by nitrocellulose filter experiments which show that this mutant has a lower affinity toward oligodeoxynucleotide substrates than the other DNA binding site mutants and a much lower affinity than wild-type EcoRI. While these and previous data clearly demonstrate that Glu144, Arg145, and Arg200 are essential for efficient substrate binding and catalysis, their involvement in the specific recognition is more intricate than proposed on the basis of the X-ray structure analysis. Our results also suggest that the specificity of EcoRI cannot easily be relaxed, and in particular not without affecting its activity.