Loverix S, Winqvist A, Strömberg R, Steyaert J
Dienst Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit, Brussel, Belgium.
Chem Biol. 2000 Aug;7(8):651-8. doi: 10.1016/s1074-5521(00)00005-3.
The microscopic events of ribonuclease (RNase) catalyzed phosphoryl transfer reactions are still a matter of debate in which the contenders adhere to either the classical concerted acid-base mechanism or a more sequential triester-like mechanism. In the case of RNase A, small thio-effects of the nonbridging oxygens have been invoked in favor of the classical mechanism. However, the RNase T1 catalyzed transphosphorylation of phosphorothioate RNA is highly stereoselective. R(P) thio-substituted RNA is depolymerized 60000 times faster than S(P) thio-substituted RNA by this enzyme, whereas the uncatalyzed cleavage of both substrates occurs at comparable rates. We combined site-directed mutagenesis in the RNase active site and stereospecific thio-substitution of an RNA substrate to probe the intermolecular interactions of the enzyme with the nonbridging pro-S(P) oxygen that bring about this stereoselectivity of RNase T1.
Thio-substitution of the nonbridging pro-S(P) oxygen in the substrate afflicts chemical turnover but not ground state binding whereas thio-substitution of the nonbridging pro-R(P) oxygen does not affect the kinetics of RNase T1. Site-directed mutagenesis of the catalytic base Glu58 impairs the enzyme's ability to discriminate both phosphorothioate diastereomers. Glu58Ala RNase T1 cleaves R(P) and S(P) phosphorothioate RNA with similar rates. The dependence of the pro-S(P) thio-effect on the presence of the Glu58 carboxylate evidences a strong rate-limiting interaction between the nonbridging pro-S(P) oxygen and the catalytic base Glu58 in the wild type enzyme.
Based on these results, we put forward a new triester-like mechanism for the RNase T1 catalyzed reaction that involves a three-centered hydrogen bond between the 2'-OH group, the nonbridging pro-S(P) oxygen and one of the carboxylate oxygens of Glu58. This interaction allows nucleophilic attack on an activated phosphate to occur simultaneously with general base catalysis, ensuring concerted phosphoryl transfer via a triester-like mechanism.
核糖核酸酶（RNase）催化的磷酰基转移反应的微观过程仍存在争议，争论双方分别支持经典的酸碱协同机制或更具顺序性的类似三酯的机制。就核糖核酸酶A而言，非桥连氧原子的小硫效应被用来支持经典机制。然而，核糖核酸酶T1催化的硫代磷酸酯RNA的转磷酸化反应具有高度的立体选择性。该酶催化R(P)硫代取代的RNA解聚的速度比S(P)硫代取代的RNA快60000倍，而两种底物的非催化裂解速率相当。我们将核糖核酸酶活性位点的定点诱变与RNA底物的立体特异性硫代取代相结合，以探究酶与非桥连的前S(P)氧原子之间的分子间相互作用，正是这种相互作用导致了核糖核酸酶T1的这种立体选择性。
底物中非桥连的前S(P)氧原子的硫代取代影响化学周转，但不影响基态结合，而非桥连的前R(P)氧原子的硫代取代不影响核糖核酸酶T1的动力学。催化碱基Glu58的定点诱变损害了该酶区分两种硫代磷酸酯非对映体的能力。Glu58Ala核糖核酸酶T1以相似的速率切割R(P)和S(P)硫代磷酸酯RNA。前S(P)硫效应依赖于Glu58羧酸盐的存在，这证明了野生型酶中非桥连的前S(P)氧原子与催化碱基Glu58之间存在强烈的限速相互作用。
基于这些结果，我们提出了一种新的类似三酯的机制，用于核糖核酸酶T1催化的反应，该机制涉及2'-OH基团、非桥连的前S(P)氧原子与Glu58的一个羧酸盐氧原子之间的三中心氢键。这种相互作用使得对活化磷酸盐的亲核攻击能够与一般碱催化同时发生，确保通过类似三酯的机制进行协同磷酰基转移。
