Kinetic analysis of delta ribozyme cleavage

The ability of delta ribozyme to catalyze the cleavage of an 11-mer RNA sub strate 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 seem s to be limited by the product release step at 37 degrees C and by the chem ical cleavage step at 56 degrees C. We observed substrate inhibition at hig h concentrations of the 11-mer substrate. Cleavage rate constants were dete rmined with a structural derivative characterized by an ultrastable LA tetr aloop. 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 e xamined ribozymes. Various cleavage inhibition and gel-shift assays with an alogues, substrate, and both active and inactive ribozymes were performed. The 2'-hydroxyl group adjacent to the scissile phosphate was shown to be in volved in binding with the ribozyme, while the essential cytosine residue o f the J4/2 junction was shown to contribute to substrate association. We cl early show that substrate binding to the delta ribozyme is not restricted t o the formation of a helix located downstream of the cleavage site. Using t hese results, we postulate a kinetic pathway involving a conformational tra nsition step essential for the formation of the active ribozyme/substrate c omplex.