Publication Date:
2013-03-13
Description:
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 Cu 2+ for optimal catalysis of thiophosphoryl transfer from GTPS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu 2+ , indicating a specific catalytic role independent of any potential interactions with the GTPS thiophosphoryl group. In-line probing and ATPS competition both argue against direct Cu 2+ binding by RNA; rather, these data establish that Cu 2+ enters the active site within a Cu 2+ •GTPS or Cu 2+ •GTP chelation complex, and that Cu 2+ •nucleobase interactions further enforce Cu 2+ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg 2+ with [Co(NH 3 ) 6 ] 3+ significantly reduced product yield, but not k obs , indicating that the role of inner-sphere Mg 2+ coordination is structural rather than catalytic. Replacing Mg 2+ with alkaline earths of increasing ionic radii (Ca 2+ , Sr 2+ and Ba 2+ ) 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.
Print ISSN:
0305-1048
Electronic ISSN:
1362-4962
Topics:
Biology
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