USE OF INTRINSIC BINDING-ENERGY FOR CATALYSIS BY AN RNA ENZYME

The contribution of several individual ribozyme substrate base pairs t o binding and catalysis has been investigated using hammerhead ribozym e substrates that were truncated at their 3' or 5' ends, The base pair s at positions 1.1-2.1 and 15.2-16.2, which flank the conserved core, each contribute 10(4)-fold in the chemical step, without affecting sub strate binding, In contrast, base pairs distal to the core contribute to substrate binding but have no effect on the chemical step, These re sults suggest a ''fraying model'' in which each ribozyme-substrate hel ix can exist in either an unpaired (''open'') state or a helical (''cl osed'') state, with the closed state required for catalysis, The base pairs directly adjacent to the conserved core contribute to catalysis by allowing the closed state to form, Once the number of base pairs is sufficient to ensure that the closed helical state predominates, addi tional residues provide stabilization of the helix, and therefore incr ease binding, but have no further effect on the chemical step, Remarka bly, the > 5 kcal/mol free energy contribution to catalysis from each of the internal base pairs is considerably greater than the free energ y expected for formation of a base pair, It is suggested that this unu sually large energetic contribution arises because free energy that is typically lost in constraining residues within a base pair is express ed in the transition state, where it is used for positioning, This ext ends the concept of ''intrinsic binding energy'' from protein to RNA e nzymes, suggesting that intrinsic binding energy is a fundamental feat ure of biological catalysis.