Table 1.

Binding and Dissociation Rate Constants for 5′-Splice Site Analog Substrates

IGS-sequence: 3′GGAGGUUUGG5′	koff (min−1)	Kd (nM)	Δ ΔG (kcal/mol)
CCUCdTMe	1.5a	15b	−0.2
CCUCdTA	1.9b	19c	(0)
CCUCdTAAACC	2.5•10−4	0.0025	−5.4
CCUCdTAAAAA	0.17	1.7	−1.5

Dissociation rate constants from the E•S complex at 30 °C, pH 6.0. Equilibrium dissociation constants were calculated from the measured dissociation rate constants and the association rate constant of 1•10⁸ M⁻¹min⁻¹ for CCUCUAAACC and CCUCUAAAAA (data not shown). This association rate constant reflects the formation of the ribozyme-substrate duplex and is the same, within error, as that determined previously for the L-21 ScaI ribozyme (1, 2, 5). Deoxyribose substitution at position –1 (Chart 1) has no effect on the association rate constant (57). Values are averages of two or more independent experiments giving rate constants that were the same within 3-fold.

^aThe dissociation rate constant for CCUCdTMe is too fast to be measured accurately and was therefore calculated from the measured dissociation rate constant for CCCUCUMe, with the effect from the deletion of –6C (Chart 1), and the deoxyribose substitution at position –1 accounted for by comparing the dissociation rate constants for CCUCdTAAAAA and CCCUCdTAAAAA (for –6C) and CCCUCdTA and CCCUCUA (for the –1 deoxyribose).

^bThe dissociation rate constant for CCUCdTA is too fast to be measured accurately and was therefore calculated from the dissociation constant for CCCUCdTA accounting for the effect from the –6 base pair as described in footnote a.