The effects of temperature, pH, and magnesium ion concentration on the internal equilibrium of the hammerhead ribozyme reaction were determined in order to better understand why the ribozyme-bound substrate RNA is 99% cleaved at equilibrium. Cleavage of substrate is more efficient at higher temperatures because a large entropy gain upon cleavage outweighs an enthalpically unfavorable generation of a 2',3'-cyclic phosphate product. The delta H of the reaction is as expected from bond energies, and provides no indication of high-energy ribozyme/substrate interactions that are lost upon cleavage. The rate constants of both cleavage and ligation increase log-linearly with pH between 5.6 and 8.0, indicating that a deprotonation step is required for both cleavage and ligation. The magnesium ion dependence of the internal equilibrium suggests that either the number or the affinity of bound magnesium ions changes upon cleavage. Since the very slow rate of hydrolysis of the 2',3'-cyclic terminus of product P1 was unaffected by the presence of the ribozyme, we conclude that hydrolysis is not a significant side reaction of the hammerhead cleavage reaction.
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