Two kinetically well-characterized hammerheads with different arm lengths were used to reinvestigate the cleavage properties of substrates with the four natural nucleotides at position 17, the residue 5' to the cleavage site. From experiments measuring substrate binding affinity, cleavage rates, and the internal equilibrium, free energy profiles of the reaction of all four substrates were constructed. Each nucleotide at the cleavage site affects the energy profile quite differently. Whereas C and U have the saline ground state energy, U destabilizes the transition state by 1 kcal/mol. A destabilizes both the ground and transition states by 1 kcal/mol, and G stabilizes the ground state by 2 kcal/mol and destabilizes the transition state by 4 kcal/mol. These data, along with experiments with the C3U mutant hammerhead, indicate that although an N3-N17 pair can form, the contribution to the binding energy for the wild-type (C3-C17) hammerhead is quite small. Thus, the energetic cost of disrupting the C3-C17 pair is not great, consistent with several proposals that this occurs during cleavage. The data also suggest that the structure in the transition state involves different stabilizing interactions with nucleotide 17 than those that are observed in the ground state. Finally, the A17 hammerhead may cleave by a slightly different reaction pathway.
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