Cobalt hexammine inhibition of the hammerhead ribozyme
Cobalt hexammine inhibition of the hammerhead ribozyme
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DOI：
10.1021/bi001141g
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发表时间：
2000-09-19
期刊：
影响因子：
2.9
通讯作者：
DeRose, VJ
中科院分区：
文献类型：
--
作者：
Horton, TE;DeRose, VJ
本文采用活性测定、电子顺磁共振（EPR）、圆二色谱（CD）和热变性研究等方法，研究了Co（NH_3）_6 ~（3+）对锤头状核酶的影响。在500 μ M Mn ~（2+）（0.1 M NaCl）溶液中，Co（NH_3）（6）（3+）能有效地置换与核酶结合的Mn ~（2+），其表观解离常数为K-dapp = 22 +/- 4.2 μ M。Mn 2+的置换与Co（NH 3）（6）（3+）在500 μ M Mn 2+中对锤头活性的抑制一致，使WT锤头的活性降低了约15倍，抑制常数Ki = 30.9 +/-2.3 μ M。在Co（NH_3）（6）（3+）和低浓度Mn ~（2+）存在下观察到残余的“缓慢”活性。在这些条件下，单个Mn 2+离子保持结合，并且具有与先前在1 M NaCl中对锤头状核酶中的最高亲和力Mn 2+位点观察到的相同的低温EPR谱，暂时归因于A9/G10.1位点[Morrissey，S. R.，Horton，T. E、和DeRose，V.J.（2000）J.Am. 122，3473-3481]。圆二色性和热变性实验还揭示了伴随观察到的由添加Co（NH3）（6）（3+）诱导的裂解和Mn 2+置换的抑制的结构效应。综上所述，数据表明，高亲和力CO（NH 3）（6）（3+）网站是负责显着抑制伴随着锤头状核酶的结构变化。此外，结果支持一个模型，其中至少有两种类型的金属网站，其中之一需要内球协调，支持锤头活动。
The effects of Co(NH3)(6)(3+) On the hammerhead ribozyme are analyzed using several techniques, including activity measurements, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies and thermal denaturation studies. Co(NH3)(6)(3+) efficiently displaces Mn2+ bound to the ribozyme with an apparent dissociation constant of K-d app = 22 +/- 4.2 mu M in 500 mu M Mn2+ (0.1 M NaCl). Displacement of Mn2+ coincides with Co(NH3)(6)(3+) inhibition of hammerhead activity in 500 mu M Mn2+, reducing the activity of the WT hammerhead by similar to 15-fold with an inhibition constant of K-i = 30.9 +/- 2.3 mu M. A residual 'slow' activity is observed in the presence of Co(NH3)(6)(3+) and low concentrations of Mn2+. Under these conditions, a single Mn2+ ion remains bound and has a low-temperature EPR spectrum identical to that observed previously for the highest affinity Mn2+ site in the hammerhead ribozyme in 1 M NaCl, tentatively attributed to the A9/G10.1 site [Morrissey, S. R., Horton, T. E., and DeRose, V. J. (2000) J. Am. Chem. Soc. 122, 3473-3481]. Circular dichroism and thermal denaturation experiments also reveal structural effects that accompany the observed inhibition of cleavage and Mn2+ displacement induced by addition of Co(NH3)(6)(3+). Taken together, the data indicate that a high-affinity CO(NH3)(6)(3+) site is responsible for significant inhibition accompanied by structural changes in the hammerhead ribozyme. In addition, the results support a model in which at least two types of metal sites, one of which requires inner-sphere coordination, support hammerhead activity.