Kinetic and stoichiometric analysis for the binding of Escherichia coli ribonuclease HI to RNA-DNA hybrids using surface plasmon resonances
Kinetic and stoichiometric analysis for the binding of Escherichia coli ribonuclease HI to RNA-DNA hybrids using surface plasmon resonances
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DOI：
10.1074/jbc.272.35.22015
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发表时间：
1997-08-29
影响因子：
4.8
通讯作者：
Crouch, RJ
中科院分区：
文献类型：
--
作者：
Haruki, M;Noguchi, E;Crouch, RJ
为了理解核糖核酸酶H如何识别RNA-DNA杂合体底物，我们使用表面等离子体共振（BIAcore（TM））分析了大肠杆菌RNase HI与长度范围从18至36个碱基对（bp）的RNA-DNA杂合体结合的动力学参数。该酶与36 bp底物结合的k（on）和k（off）值分别为1.5 × 10（6）M-1 s（-1）和3.2 × 10（-2）s（-1）。用较短的底物得到类似的值。在不存在Mg 2+的情况下，h（开）和h（关）的值分别为2.1 x 10（5）M-1 s（-1）和1.3 x 10（-1）s（-1），在存在Mg 2+的情况下，h（开）和h（关）的值进一步降低至7.4 x 10（3）M-1 s（-1）和2.6 x 10（-2）s（-1）。动力学参数类似于野生型酶，获得使用活性位点突变酶，天冬氨酸（134）取代丙氨酸，而大大降低的on-rate观察到另一种无活性的突变酶，其中的基本突起被消除，从而区分不良的催化和不能结合到基板。RNase HI与18、24、30和36 bp底物结合的化学计量分析与以前的报告一致，表明RNase HI与9-10 bp的RNA-DNA杂合体结合。
To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding of Escherichia coli RNase HI to RNA-DNA hybrids ranging in length from 18 to 36 base pairs (bp) using surface plasmon resonance (BIAcore((TM))). The k(on) and k(off) values for the binding of the enzyme to the 36 bp substrate were 1.5 x 10(6) M-1 s(-1) and 3.2 x 10(-2) s(-1), respectively, Similar values were obtained with the shorter substrates, Using uncleavable 2'-O-methylated RNA-DNA substrates, values for h(on) and h(off) were 2.1 x 10(5) M-1 s(-1) and 1.3 x 10(-1) s(-1) in the absence of Mg2+ that were further reduced in the presence of Mg2+ to 7.4 x 10(3) M-1 s(-1) and 2.6 x 10(-2) s(-1). Kinetic parameters similar to the wild-type enzyme were obtained using an active-site mutant enzyme, Asp(134) replaced by Ala, whereas a greatly reduced on-rate was observed for another inactive mutant enzyme, in which the basic protrusion is eliminated, thereby distinguishing between poor catalysis and inability to bind to the substrate. Stoichiometric analyses of RNase HI binding to substrates of 18, 24, 30, and 36 bp are consistent with previous reports suggesting that RNase HI binds to 9-10 bp of RNA DNA hybrid.