Probing the Tetrahymena group I ribozyme reaction in both directions.
The Tetrahymena L-21 ScaI ribozyme derived from the self-splicing group I intron catalyzes a reversible reaction analogous to the first step of self-splicing: CCCUCUA (S) + [UC]G right harpoon over left harpoon CCCUCU (P) + [UC]GA. To relate our understanding of the ribozyme to the self-splicing reaction and to further the mechanistic dissection of the ribozyme reaction, we have established a quantitative kinetic and thermodynamic framework for the forward and reverse reaction of the L-21 ScaI ribozyme under identical conditions. Examination of the framework shows that binding of products is cooperative with binding enhanced 5-fold, as was observed previously for binding of the substrates. Further, binding of UCGA is 12-fold weaker than binding of the unphosphorylated UCG, analogous to the 20-fold weaker binding of phosphorylated S relative to P; the molecular interactions underlying the stronger binding of UCG were traced to the 3'-hydroxyl group of UCG. The symmetrical effects on binding of substrates and products result in the equilibrium between ribozyme-bound species, K(int), that is essentially unperturbed from the solution equilibrium, K(ext) (K(int) = [E.P.UCGA]/[E.S.UCG] = 4.6 and K(ext) = [P][UCGA]/[S][UCG] = 1.9). Last, we show that the pK(a) values of the nucleophiles in the forward and reverse reactions are >/=10. This observation suggests that metal ion activation of the nucleophile and stabilization of the leaving group can only account for a portion of the rate enhancement of this ribozyme. These and prior results suggest that the Tetrahymena group I ribozyme, analogous to protein enzymes, uses multiple catalytic strategies to achieve its large rate enhancement.
Karbstein
Katrin
K
Department of Biochemistry, Stanford University, Beckman Center B400, Stanford, California 94305-5307, USA. herschla@cmgm.stanford.edu
Carroll
Kate S
KS
Herschlag
Daniel
D
D000818:Animals; D001665:Binding Sites; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006878:Hydroxides / Q000737:chemistry; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry*; D016053:RNA, Protozoan / Q000737:chemistry; D013379:Substrate Specificity; D013768:Tetrahymena / Q000201:enzymology*; D013816:Thermodynamics
C116167:GIR1 ribozyme; D006878:Hydroxides; D008670:Metals; D016337:RNA, Catalytic; D016053:RNA, Protozoan; C031356:hydroxide ion