KINETICS AT A MULTIFUNCTIONAL RNA ACTIVE-SITE

Kinetics of a self-capping RNA, Iso6, have been investigated to constr ain the catalytic mechanism. The role of phosphates has been examined by varying the number of phosphates on the nucleophilic attacking grou p or on the RNA. While the number of phosphates in the nucleophile aff ects capping kinetics, only K-M but not k(cat) is altered. The K-M val ues for GMP, GDP, GTP and ppppG are 200, 11, 13 and 31 mu M, respectiv ely. A reaction product, pyrophosphate, is also found to strongly inhi bit RNA activities through a competitive exchange mechanism with an ap parent K-i of 200 nM. Uniquely strong binding of pyrophosphate support s the idea that capping originated by utilization of the initial pyrop hosphate leaving group site for capping nucleophiles. In contrast to t he nucleophile phosphate, change of 5' RNA terminus from triphosphate to tetraphosphate enhances the overall rate and k(cat) by 40%, with li ttle effect on K-M. Thus, only the leaving group appears to affect the rate of the chemical transformation. We propose two possible mechanis ms that explain this apparent rate-limiting chemical step, either diss ociation of pyrophosphate to form a metaphosphate monoester intermedia te or formation of a circular phosphoramidate intermediate, using an i nternal RNA nitrogenous group. A single essential Ca ion is required f or all activities. (C) 1998 Academic Press.