Site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease I
Site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease I
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DOI：
10.1006/jmbi.1996.0703
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发表时间：
1996-12-20
影响因子：
5.6
通讯作者：
Connolly, BA
中科院分区：
文献类型：
--
作者：
Jones, SJ;Worrall, AF;Connolly, BA
牛胰腺脱氧核糖核酸酶I （DNase I）是一种特性良好的内切酶，它可以切割双链DNA以产生5'磷酸化的多核苷酸。具有两种不同寡核苷酸的dna酶I的go晶体结构显示，在可裂磷酸盐附近存在几个残基（R9, E78, H134, D168， D212和H252）。这些氨基酸在催化机制中所起的作用已经用定点诱变进行了研究。使用了以下改型：R9A、E78T、H134Q、D168S、D212S和H252Q。研究了所有6个具有DNA和一个小的显色底物胸腺嘧啶-3',5'-二（对硝基苯）-磷酸的突变体的动力学。只有R9A和E78T显示。两个底物的任何显著的转换。D168S、H134Q、D212S和H252Q对DNA的活性极低，对胸腺嘧啶-3′，5′-二-（对硝基苯）-磷酸没有活性。这些结果表明，H134、D168、D212和H252在催化机理中起关键作用。说明H134和H252（分别与E78和D212形成氢键）具有一般的酸催化作用和一般的碱催化作用。DNase I也需要Mg2+， E39已被确定为该金属离子的配体。我们提出D168作为第二个Mg2+的配体，因此dna酶I使用双金属离子水解机制。两种镁离子都用于提供亲电催化。角色分配是基于突变结果、结构信息、不同物种dna酶I之间的同源性以及与外切酶III的比较。然而，明确地为每个氨基酸/金属离子指定特定的催化作用仍然是不可行的。(C) 1996学术出版社有限公司
Bovine pancreatic deoxyribonuclease I (DNase I) is a well characterised endonuclease which cleaves double-stranded DNA to yield 5' phosphorylated polynucleotides. Go-crystal structures of DNase I with two different oligonucleotides have revealed the presence of several residues (R9, E78, H134, D168, D212 and H252) close to the scissile phosphate. The roles that these amino acids play in the catalytic mechanism have been investigated using site-directed mutagenesis. The following variants were used: R9A, E78T, H134Q, D168S, D212S and H252Q. The kinetics of all six mutants with both DNA and a small chromophoric substrate, thymidine-3',5'-di(p-nitrophenyl)-phosphate, were studied. Only R9A and E78T showed. any significant turnover of the two substrates. D168S, H134Q, D212S and H252Q showed vanishingly low activities towards DNA and no detectable activity with thymidine-3',5'-di-(p-nitrophenyl)-phosphate. These results demonstrate that H134, D168, D212 and H252 play a critical role in the catalytic mechanism. It is suggested that H134 and H252 (which are hydrogen-bonded to E78 and D212, respectively) provided general acid and general base catalysis. DNase I also requires Mg2+ and E39 has been identified as a ligand for this metal ion. We propose that D168 serves as a ligand for a second Mg2+, and thus DNase I, uses a two metal-ion hydrolytic mechanism. Both magnesium ions are used to supply electrophilic catalysis. Role assignment is based on the mutagenesis results, structural information, homologies between DNase I from different species and a comparison with exonuclease III. However, it is still not feasible to unequivocally assign a particular catalytic role to each amino acid/metal ion. (C) 1996 Academic Press Limited