Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu²⁺ for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu²⁺, indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu²⁺ binding by RNA; rather, these data establish that Cu²⁺ enters the active site within a Cu²⁺•GTPγS or Cu²⁺•GTP chelation complex, and that Cu²⁺•nucleobase interactions further enforce Cu²⁺ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg²⁺ with [Co(NH₃)₆]³⁺ significantly reduced product yield, but not k_obs, indicating that the role of inner-sphere Mg²⁺ coordination is structural rather than catalytic. Replacing Mg²⁺ with alkaline earths of increasing ionic radii (Ca²⁺, Sr²⁺ and Ba²⁺) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.