CATALYSIS OF RNA CLEAVAGE BY THE TETRAHYMENA-THERMOPHILA RIBOZYME .1. KINETIC DESCRIPTION OF THE REACTION OF AN RNA SUBSTRATE COMPLEMENTARY TO THE ACTIVE-SITE
CATALYSIS OF RNA CLEAVAGE BY THE TETRAHYMENA-THERMOPHILA RIBOZYME .1. KINETIC DESCRIPTION OF THE REACTION OF AN RNA SUBSTRATE COMPLEMENTARY TO THE ACTIVE-SITE
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DOI：
10.1021/bi00496a003
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发表时间：
1990-11-06
期刊：
影响因子：
2.9
通讯作者：
CECH, TR
中科院分区：
文献类型：
--
作者：
HERSCHLAG, D;CECH, TR
来源于四膜虫前核糖体RNA的间插序列（IVS）的核酶催化位点特异性内切核酸酶反应：G2 CCCUCUA 5 + G. dblarw。G2 CCCUCU + GA 5（G =鸟苷）。该反应类似于前体rRNA自我剪接的第一步，产物G2 CCCUCU类似于5“-外显子。从在50 ℃下的预稳态和稳态动力学测量中得出以下机理结论。在10 nM Mg 2+的存在下，在10 ℃和中性pH下进行。kcat/Km的值= 9 ×对于具有饱和G的寡核苷酸底物，107 M-1 min-1表示限速结合。结合的该速率常数具有预期形成RNA的量级。寡核苷酸之间的RNA双链体。（系统发育和突变分析表明，该底物通过与IVS内的互补序列进行碱基配对来识别）。kcat = 0.1 min-1的值代表5“-外显子类似物G2 CCCUCU的限速解离。由于G2 CCCUCU的这种缓慢解离速率，产物GA 5首先从核酶解离。产物G2 CCCUCU和底物G2 CCCUCUA 5与核酶的5“外显子结合位点的类似结合，Kd = 1-2 nM，表明底物的pA 5部分对结合没有净贡献。底物和产物都结合。104-比从与5“-外显子结合位点的碱基配对所预期的强1倍（6 kcal/mol）。因此，三级相互作用参与结合。G2 CCCUCU的结合和G的结合是独立的。这些和其他数据表明，寡核苷酸底物G2 CCCUCUA 5的结合和G的结合基本上是随机和独立的。三元络合物的反应速率常数计算为kc·simeq。350 min-1，该速率常数未反映在具有饱和G.采用最简单的解释，其中kc表示化学步骤的速率。观察到在不存在G的情况下通过四膜虫核酶的位点特异性内切核酸酶反应;用溶剂替换鸟苷的化学步骤的速率kc（-G）= 0.7min-1，为500-比饱和鸟苷慢一倍。kcat/Km的值= 6 ×107 M-1 min-1的水解反应，仅略小于饱和鸟苷，因为在这两种情况下，寡核苷酸底物的结合主要是限速的。这种核酶接近蛋白酶的kcat/Km极限值，可以认为已经实现了“催化完美”。
A ribozyme derived from the intervening sequence (IVS) of the Tetrahymena preribosomal RNA catalyzes a site-specific endonuclease reaction: G2CCCUCUA5 + G .dblarw. G2CCCUCU + GA5 (G = guanosine). This reaction is analogous to the first step in self-splicing of the pre-rRNA, with the product G2CCCUCU analogous to the 5''-exon. The following mechanistic conclusions have been derived from pre-steady-state and steady-state kinetic measurements at 50.degree. C and neutral pH in the presence of 10 nM Mg2+. The value of kcat/Km = 9 .times. 107 M-1 min-1 for the oligonucleotide substrate with saturating G represents rate-limiting binding. This rate constant for binding is of the order expected for formation of a RNA .cntdot. RNA duplex between oligonucleotides. (Phylogenetic and mutational analyses have shown that this substrate is recognized by base pairing to a complementary sequence within the IVS). The value of kcat = 0.1 min-1 represents rate-limiting dissociation of the 5''-exon analogue, G2CCCUCU. The product GA5 dissociates first from the ribozyme because of this slow off-rate for G2CCCUCU. The similar binding of the product, G2CCCUCU, and the substrate, G2CCCUCUA5, to the 5''exon binding site of the ribozyme, with Kd = 1-2 nM, shows that the pA5 portion of the substrate makes no net contribution to binding. Both the substrate and product bind .apprx. 104-fold (6 kcal/mol) stronger than expected from base pairing with the 5''-exon binding site. Thus, tertiary interactions are involved in binding. Binding of G2CCCUCU and binding of G are independent. These and other data suggest that binding of the oligonucleotide substrate, G2CCCUCUA5, and binding of G are essentially random and independent. The rate constant for reaction of the ternary complex is calculated to be kc .simeq. 350 min-1, a rate constant that is not reflected in the steady-state rate parameters with saturating G. The simplest interpretation is adopted, in which kc represents the rate of the chemical step. A site-specific endonuclease reaction by the Tetrahymena ribozyme in the absence of G was observed; the rate of the chemical step with solvent replacing guanosine, kc(-G) = 0.7 min-1, is .apprx. 500-fold slower than that with saturating guanosine. The value of kcat/Km = 6 .times. 107 M-1 min-1 for this hydrolysis reaction is only slightly smaller than that with saturating guanosine, because the binding of the oligonucleotide substrate is predominantly rate-limiting in both cases. This ribozyme, which approaches the limiting values of kcat/Km for protein enzymes, can be considered to have achieved "catalytic perfection".