Temperature‐dependent change in the rate‐determining step in a reaction catalyzed by a hammerhead ribozyme

Abstract

To characterize the reaction catalyzed by a hammerhead ribozyme, the dependence on temperature of the reaction was examined. An Arrhenius plot revealed a transition that indicated a temperature-dependent change in the activation energy at around 25 degrees C. Thermodynamic parameters of the reaction were estimated at 10 and 35 degrees C. The analyses led to the following conclusions. At 25-50 degrees C, the chemical cleavage step (kcleav) was the rate-determining step, and the cleaved fragments dissociated from the ribozyme at a higher rate than the rate of the chemical reaction. When the temperature was below 25 degrees C, the cleaved fragments adhered to the ribozyme more tightly and the product dissociation step became the rate-determining step. Above 50 degrees C, the rate of the reaction decreased because, at such high temperatures, the formation of the Michaelis-Menten complex (duplex formation) was hampered by thermal melting. A conformational change in the ribozyme-substrate complex was not the rate-determining step at any of the temperatures examined.