Herschlag D, Eckstein F, Cech T R
Department of Biochemistry, Beckman Center, Stanford University, California 94305-5307.
Biochemistry. 1993 Aug 17;32(32):8312-21. doi: 10.1021/bi00083a035.
The ribozyme derived from the intron of Tetrahymena thermophila pre-rRNA catalyzes a site-specific endonuclease reaction with both RNA and DNA oligonucleotides. The total transition-state stabilization by the ribozyme, encompassing the binding and chemical steps, is 4.8 kcal/mol greater with a single ribose at the cleavage site relative to the all-deoxyribose substrate. Here we show that this effect is specific to the chemical transition state, with a contribution of only approximately 0.7 kcal/mol toward binding. Substrates with a series of 2'-substituents, -OH(ribo), -F2 (2',2'-difluoro-2'-deoxyribo), F(2'-fluoro-2'-deoxyribo), and -H(deoxyribo), follow a linear free energy relationship between the rate of the chemical step of the ribozyme-catalyzed reaction and the pK(a) of the leaving group, with slope beta leaving group approximately -0.8. Because proton donation to the 3'-oxygen atom from a general acid of the ribozyme would be expected to render the rate insensitive to the pK(a) of the leaving group, it is suggested that this ribozyme does not employ general acid catalysis. The 2'-OCH3 (2'-methoxy-2'-deoxyribo) substituent does not follow this correlation, apparently due to steric hindrance within the active site. The rate of cleavage of the 2'-substituted substrates by the ribozyme follows the order 2'-F2 > -F > -H, suggestive of an inductive effect, i.e., acceleration of the reaction by electron-withdrawing groups. The 2'-OH group provides the largest transition-state stabilization. Because of uncertainty in the relative effect of the 2'-OH and 2'-H substituents on the pK(a) of the neighboring 3'-oxygen leaving group, we do not discount the possibility of interactions between the 2'-hydroxyl group and the ribozyme that further enhance reactivity. Nevertheless, the 2'-OH effect can be explained at least partially by an intramolecular hydrogen bond to an incipient oxyanion at the neighboring 3'-position. This oxyanion is forming as the phosphodiester bond is breaking, explaining why the stabilization is specific to the transition state. Analogous differential hydrogen bonding might be widely used by enzymes to achieve selective transition-state stabilization.
源自嗜热四膜虫前体rRNA内含子的核酶催化RNA和DNA寡核苷酸的位点特异性内切核酸酶反应。相对于全脱氧核糖底物，核酶在切割位点具有单个核糖时，包括结合步骤和化学步骤在内的总过渡态稳定化作用比其高4.8千卡/摩尔。在此我们表明，这种效应是化学过渡态所特有的，对结合的贡献仅约为0.7千卡/摩尔。具有一系列2'-取代基（-OH（核糖）、-F2（2',2'-二氟-2'-脱氧核糖）、F（2'-氟-2'-脱氧核糖）和-H（脱氧核糖））的底物，在核酶催化反应的化学步骤速率与离去基团的pK(a)之间呈现线性自由能关系，斜率β离去基团约为-0.8。由于预期核酶的一般酸向3'-氧原子的质子供体作用会使反应速率对离去基团的pK(a)不敏感，因此表明该核酶不采用一般酸催化。2'-OCH3（2'-甲氧基-2'-脱氧核糖）取代基不遵循这种相关性，显然是由于活性位点内的空间位阻。核酶对2'-取代底物的切割速率遵循2'-F2 > -F > -H的顺序，表明存在诱导效应，即吸电子基团加速反应。2'-OH基团提供最大的过渡态稳定化作用。由于2'-OH和2'-H取代基对相邻3'-氧离去基团的pK(a)的相对影响存在不确定性，我们不排除2'-羟基与核酶之间相互作用进一步增强反应性的可能性。然而，2'-OH效应至少可以部分通过与相邻3'-位置的初始氧阴离子形成分子内氢键来解释。这种氧阴离子是在磷酸二酯键断裂时形成 的，这就解释了为什么这种稳定化作用是过渡态所特有的。类似的差异氢键作用可能被酶广泛用于实现选择性的过渡态稳定化。
