A POSITIVE ENTROPY CHANGE FOR GUANOSINE BINDING AND FOR THE CHEMICAL STEP IN THE TETRAHYMENA RIBOZYME REACTION

The ribozyme derived from the group I intron of Tetrahymena thermophil a binds an exogenous guanosine nucleotide, which acts as the nucleophi le in the sequence-specific cleavage of oligonucleotides. By examining the temperature dependence of the reaction under conditions where K-m = K-d, we conclude the following: (1) Guanosine 5'-monophosphate (pG) binds to the closed ribozyme-oligonucleotide substrate complex with a positive entropy change (Delta S degrees' = +23 eu) and an enthalpy c hange (Delta H degrees') close to zero. This is contrary to the expect ation that binding would cause increased order (negative Delta S degre es) and be driven by a negative Delta H degrees. (2) Inosine and 2-ami nopurine riboside, each lacking two hydrogen-bonding moieties relative to guanosine, also bind with a positive entropy value and an unfavora ble (positive) Delta H degrees'. From this result, we suggest that the hydrogen-bonding moieties make an enthalpic contribution to guanosine binding overcoming an intrinsic unfavorable Delta H. (3) At 0 degrees C, there is equally tight binding of pG in the presence and absence o f oligonucleotide substrate bound to the ribozyme. Thus, energetic int eractions responsible for the thermodynamic coupling between pG and ol igonucleotide substrate binding seen at higher temperatures are indire ct. (4) The activation barrier of the chemical step is stabilized by a positive Delta S-double dagger (+31 to 39 eu). This stabilization is seen in four reactions using substrates with two different leaving gro ups in the presence and absence of pG, suggesting that the entropic co ntribution is inherent to the active site. The positive Delta S values for the chemical step and for the binding of pG can be explained by a conformational change or release of water. Thus, although hydrogen bo nding contributes to binding of nucleotides to this RNA enzyme as prev iously thought, it is these other events which produce a positive Delt a S that provide the energetic driving force for binding.