Profenno L A, Kierzek R, Testa S M, Turner D H
Department of Chemistry, Box 270216, University of Rochester, Rochester, New York 14627-0216, USA.
Biochemistry. 1997 Oct 14;36(41):12477-85. doi: 10.1021/bi9708895.
The dynamics of binding of various guanosine, or G, substrates to the Tetrahymena thermophila L-21 ScaI ribozyme have been investigated by fluorescence-detected stopped-flow experiments. Upon rapid mixing of various G substrates with a preformed complex of the ribozyme and the fluorescent 5' splice site analogue CCUCUepsilonA, fluorescence transients that provide rates for binding of G substrates and the rate-limiting step for transesterification are observed. The measured apparent bimolecular rate constant for binding of pG is 10(3) M-1 s-1, much slower than expected for diffusion. pG appears to bind to the preformed complex of the ribozyme and CCUCUepsilonA in at least two steps, a bimolecular step followed by at least one conformational change. This two-step binding of pG, involving a rapid pre-equilibrium, leads to the slow apparent rate constant for binding of pG. Furthermore, the 2'-OH of pG and of the 3' terminal G of the G substrate GUCG and the nonbridging pro-Sp phosphoryl oxygen atom at the site of phosphoryl transfer on CCUCUepsilonA appear to mediate formation of a properly conformed docked ternary complex of the G substrate, 5' splice site, and ribozyme which may represent an intermediate required for initiation of transesterification. It is possible that the 2'-OH of pG and this nonbridging pro-Sp phosphoryl oxygen interact, directly or indirectly, with one another.
通过荧光检测的停流实验，研究了各种鸟苷（或G）底物与嗜热四膜虫L - 21 ScaI核酶的结合动力学。将各种G底物与核酶和荧光5'剪接位点类似物CCUCUεA的预形成复合物快速混合后，观察到荧光瞬变，其提供了G底物的结合速率和酯交换的限速步骤。测得的pG结合的表观二级速率常数为10³ M⁻¹ s⁻¹，比扩散预期的要慢得多。pG似乎至少通过两步与核酶和CCUCUεA的预形成复合物结合，第一步是双分子步骤，随后至少发生一次构象变化。pG的这种两步结合涉及快速的预平衡，导致pG结合的表观速率常数较慢。此外，pG的2'-OH、G底物GUCG的3'末端G的2'-OH以及CCUCUεA上磷酸转移位点的非桥连亲核磷酰氧原子似乎介导了G底物、5'剪接位点和核酶形成正确构象的对接三元复合物，这可能代表酯交换起始所需的中间体。pG的2'-OH与这个非桥连亲核磷酰氧可能直接或间接相互作用。
