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.