Direct demonstration of the catalytic role of binding interactions in an enzymatic reaction.

It has been suggested that the fundamental feature that distinguishes enzymes from simple chemical catalysts is the ability of enzymes to use binding interactions for catalysis. Results with the Tetrahymena group I RNA enzyme described herein directly demonstrate the catalytic contributions of binding interactions. With wild-type ribozyme, specific functional groups at a distance from the site of chemical transformation facilitate substrate binding without accelerating reaction of bound substrate; with modified ribozymes, these functional groups provide the same overall energetic effect but instead accelerate reaction of bound substrate without increasing binding. These observations are quantitatively described by a structural framework that was established by previous results. The P1 duplex between the substrate and the ribozyme's recognition sequence exists in two states, the open complex, in which the substrate is localized to the ribozyme solely by base-pairing interactions, or the closed complex, in which the duplex is docked into tertiary interactions and positioned with respect to the catalytic groups in the active site. In the absence of sufficient binding energy to ensure stable docking in the ground state, added P1 functional groups accelerate reaction of the bound substrate by helping to overcome the energetic barrier for docking into the reactive, closed complex. When the functional groups present on the P1 duplex are sufficient to ensure stable docking in the closed complex, added functional groups give stronger binding without accelerating reaction of the bound substrate. This behavior is a manifestation of the inextricable link between binding interactions and catalysis. The conclusions also have implications for interpreting the effects of site-directed mutagenesis and for the evolution of active site interactions.