Herschlag D, Piccirilli J A, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Biochemistry. 1991 May 21;30(20):4844-54. doi: 10.1021/bi00234a003.
The L-21 ScaI ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a guanosine-dependent endonuclease reaction that is analogous to the first step in self-splicing of this intervening sequence. We now describe pre-steady-state kinetic experiments, with sulfur substituting for the pro-RP (nonbridging) phosphoryl oxygen atom at the site of cleavage, that test aspects of a kinetic model proposed for the ribozyme reaction (Herschlag, D., & Cech, T. R. (1990) Biochemistry 29, 10159-10171). Thio substitution does not affect the reaction with subsaturating oligonucleotide substrate and saturating guanosine ((kcat/Km)S), consistent with the previous finding that binding of the oligonucleotide substrate limits this rate constant. In contrast, there is a significant decrease in the rate of single-turnover reactions of ribozyme-bound (i.e., saturating) oligonucleotide substrate upon thio substitution, with decreases of 2.3-fold for the reaction with guanosine ((kcat/Km)G) and 7-fold for hydrolysis [i.e., with solvent replacing guanosine; kc(-G)]. These "thio effects" are consistent with rate-limiting chemistry, as shown by comparison with model reactions. Nonenzymatic nucleophilic substitution reactions of the phosphate diester, methyl 2,4-dinitrophenyl phosphate monoanion, are slowed 4-11-fold by thio substitution for reactions with hydroxide ion, formate ion, fluoride ion, pyridine, and nicotinamide. In addition, we have confirmed that thio substitution has no effect on the nonenzymatic alkaline cleavage of RNA (Burgers, P. M. J., & Eckstein, F. (1979) Biochemistry 18, 592-596). Considering the strong preference of Mg2+ for binding to oxygen rather than sulfur, the modest thio effect on the chemical step of the ribozyme-catalyzed reaction and the absence of a thio effect on the equilibrium constant for binding of the oligonucleotide substrate suggest that the pro-RP oxygen atom is not coordinated to Mg2+ in the E.S complex or in the transition state. General implications of thio effects in enzymatic reactions of phosphate diesters are discussed.
源自嗜热四膜虫前体rRNA间隔序列的L-21 ScaI核酶催化一种依赖鸟苷的内切核酸酶反应，该反应类似于此间隔序列自我剪接的第一步。我们现在描述了前稳态动力学实验，其中用硫取代切割位点处的前-RP（非桥连）磷酰氧原子，以检验为核酶反应提出的动力学模型的各个方面（Herschlag, D., & Cech, T. R. (1990) Biochemistry 29, 10159 - 10171）。硫取代不影响与亚饱和寡核苷酸底物和饱和鸟苷的反应（(kcat/Km)S），这与先前的发现一致，即寡核苷酸底物的结合限制了该速率常数。相比之下，硫取代后核酶结合的（即饱和的）寡核苷酸底物的单周转反应速率显著降低，与鸟苷反应（(kcat/Km)G）降低2.3倍，水解反应（即溶剂取代鸟苷；kc(-G)）降低7倍。这些“硫效应”与限速化学过程一致，如与模型反应比较所示。磷酸二酯2,4-二硝基苯磷酸甲酯单阴离子与氢氧根离子、甲酸根离子、氟离子、吡啶和烟酰胺反应时，硫取代会使非酶亲核取代反应减慢4 - 11倍。此外，我们已证实硫取代对RNA的非酶碱性切割没有影响（Burgers, P. M. J., & Eckstein, F. (1979) Biochemistry 18, 592 - 596）。考虑到Mg2+强烈倾向于与氧而非硫结合，硫对核酶催化反应化学步骤的适度影响以及对寡核苷酸底物结合平衡常数无硫效应表明，在E.S复合物或过渡态中，前-RP氧原子未与Mg2+配位。讨论了硫效应在磷酸二酯酶促反应中的一般意义。
