Monovalent cations use multiple mechanisms to resolve ribozyme misfolding

Measuring the rate constant for substrate cleavage by the Candida ribozyme Ca.L-11. (A) Kinetics for the ribozyme catalysis. The initial velocity (v₀ ) for each substrate concentration ranging from 25 nM to 1500 nM was determined from a single exponential plot of the fraction of reaction as a function of time: Y = Y_max × [1 − exp(−k_obs × X)], in which v₀ = k_obs[S]. v₀ was then plotted as a function of substrate concentration. The graph shown is a nonlinear least-squares fit to the Michaelis-Menten equation; the inserted graph is also a linear least-squares fit to a plot of 1/v₀ against 1/[substrate]. The resulting kinetic parameters are K_M = 268.3 ± 86.76 nM, V_max = 128.8 ± 17.04 nM/min, and k_cat = 8.6 ± 1.1 min⁻¹, k_cat/K_M = 3.4316 ± 0.9895 × 10⁷ mol⁻¹ min⁻¹. (B) Rate constant for substrate cleavage (k_c) measured by the prefolded ribozyme. After the ribozyme renatured in Tris-HCl and prefolded in the presence of 10 mM Mg²⁺ and substrate for 10 min, GTP was added to initiate the substrate cleavage for 0–240 sec. The rate constant k_c was 0.1195 ± 0.005986 sec⁻¹.