Table 2.

Kinetics and thermodynamic constants of full-length mutant ribozymes

	Number and description of ribozyme	kobs (min−1)1			kobs (relative)2	ΔΔGM (kcal/mol)3	KM <-->N4	ΔΔGN (kcal/mol)5
		50 °C	37 °C	25 °C
13	WT	0.075 ± 0.017	1.4 × 10−3	2.1 × 10−4	(1)	(0)	(1.1 ± 0.6) × 105	(0)
14	R14C	0.017 ± 0.004	4.0 × 10−3	5.1 × 10−5	0.22 ± 0.07	−0.95 ± 0.25	(8 ± 4) × 105	−2.1 ± 0.7
15	WT A269G/A304G	0.043	8.1 × 10−3	1.3 × 10−4	0.57	−0.36	(2.1 ± 0.9) × 106	−2.1 ± 0.7
16	R14C G269A/G304A	0.012	N.D.	N.D.	0.16	−1.2	(7 ± 4) × 104	−0.9 ± 0.9
17	WT U277C	0.041	1.5 × 10−3	2.5 × 10−4	0.53	−0.39	N.D.	N.D.
18	R14C C277U	8.2 × 10−3	1.2 × 10−4	N.D.	0.11	−1.4	N.D.	N.D.
19	WT U277C/A269G/304G	0.027	1.6 × 10−3	N.D.	0.40	−0.59	N.D.	N.D.
20	R14C C277U/G269A/G304A	6.0 × 10−3	1.3 × 10−4	N.D.	0.070	−1.7	N.D.	N.D.
21	WT U259A	0.12	N.D	N.D	1.6	0.30	N.D	N.D
22	WTU340A	0.046	1.3 × 10−3	N.D	0.61	−0.32	N.D	N.D

¹Values are rate constants for refolding of the misfolded ribozyme to the native state. For the wild-type and R14C ribozymes, values are the average and range from two independent determinations. Results from single determinations are reported for the other mutant ribozymes.

²Relative values were obtained from measurements at 50 °C.

³ΔΔG^M values reflect the change in stability of the misfolded conformation for the indicated mutant, relative to the transition state for refolding. These values were calculated from the relative refolding rates as ΔΔG^M = −RT ln (1/k_obs (relative)). Values are based on measurements at 50 °C. Similar effects of mutations were observed at 25 °C for all of the mutants for which measaurements were made at 25 °C.

⁴K_{M <-->N} values are reproduced from Table 1. These values were determined using the thermodynamic cycle of P5abc binding to the native and misfolded conformers of the corresponding P5abc-deleted ribozymes.

⁵ΔΔG^N values reflect the change in stability of the native conformation for the indicated mutant, relative to the transition state for refolding, and were calculated from ΔΔG^M and the change in K_{M <-->N} as ΔΔG^N = ΔΔG^M + (−RT ln(K_M<-->N)_Mutant/(K_M<-->N)_WT) (see Figure 3).

N.D., not determined.