Kinetics at a Multifunctional RNA Active Site

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Chemistry and Biochemistry


Kinetics of a self-capping RNA, Iso6, have been investigated to constrain the catalytic mechanism. The role of phosphates has been examined by varying the number of phosphates on the nucleophilic attacking group or on the RNA. While the number of phosphates in the nucleophile affects capping kinetics, only K-M but not k(cat) is altered. The K-M values for GMP, GDP, GTP and ppppG are 200, 11, 13 and 31 mu M, respectively. A reaction product, pyrophosphate, is also found to strongly inhibit RNA activities through a competitive exchange mechanism with an apparent K-i of 200 nM. Uniquely strong binding of pyrophosphate supports the idea that capping originated by utilization of the initial pyrophosphate leaving group site for capping nucleophiles. In contrast to the nucleophile phosphate, change of 5' RNA terminus from triphosphate to tetraphosphate enhances the overall rate and k(cat) by 40%, with little effect on K-M. Thus, only the leaving group appears to affect the rate of the chemical transformation. We propose two possible mechanisms that explain this apparent rate-limiting chemical step, either dissociation of pyrophosphate to form a metaphosphate monoester intermediate or formation of a circular phosphoramidate intermediate, using an internal RNA nitrogenous group. A single essential Ca ion is required for all activities. (C) 1998 Academic Press.

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Journal of Molecular Biology





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