Kinetic analysis of δ ribozyme cleavage

The ability of delta ribozyme to catalyze the cleavage of an 11-mer RNA substrate was examined under both single- and multiple-turnover conditions. In both cases only small differences in the kinetic parameters were observed in the presence of either magnesium or calcium as cofactor. Under multiple-turnover conditions, the catalytic efficiency of the ribozyme (k(cat)/K-M) was higher at 37 degrees C than at 56 degrees C. The cleavage reaction seems to be limited by the product release step at 37 degrees C and by the chemical cleavage step at 56 degrees C. We observed substrate inhibition at high concentrations of the 11-mer substrate. Cleavage rate constants were determined with a structural derivative characterized by an ultrastable LA tetraloop. The kinetic parameters (K-cat and K-M) and dissociation constant (K-d) were almost identical for both ribozymes, suggesting that the stability of the L4 loop has a negligible impact on the catalytic activities of the examined ribozymes. Various cleavage inhibition and gel-shift assays with analogues, substrate, and both active and inactive ribozymes were performed. The 2'-hydroxyl group adjacent to the scissile phosphate was shown to be involved in binding with the ribozyme, while the essential cytosine residue of the J4/2 junction was shown to contribute to substrate association. We clearly show that substrate binding to the delta ribozyme is not restricted to the formation of a helix located downstream of the cleavage site. Using these results, we postulate a kinetic pathway involving a conformational transition step essential for the formation of the active ribozyme/substrate complex.