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°C. Thermodynamic parameters of the reaction were estimated at 10 and 35°C. The analyses led to the following conclusions. At 25–50°C. the chemical cleavage step (k _cleav 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°C, the cleaved fragments adhered to the ribozyme more tightly and the product dissociation step became the rate‐determining step. Above 50°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.