Table 3.

Kinetics of cytosine flipping and catalytic loop motion in Trp-engineered M.HhaI

Reaction	Cognate dead-end (Figure 3): Target base pair = C:G; cofactor = AdoHcy			Mismatch dead-end (Figure 4): Target base pair = C:T; cofactor = AdoHcy			Cognate full cycle (Figure 5B): Target base pair = C:G; cofactor = AdoMet
Goodness of fit (χ2 × 105)	12.5			8.65			7.32
Parameters	Rate, s−1	Amplitude		Rate, s−1	Amplitude		Rate, s−1	Amplitude
		Abs.	Fl.		Abs.	Fl.		Abs	Fl.
Step 1	19.0 ± 1.1	0.26 ± 0.03	−0.32 ± 0.03	63 ± 2	0.53 ± 0.02	−0.56 ± 0.02	180 ± 5	0.23 ± 0.004	−0.13 ± 0.004
Step 2	6.9 ± 0.2	0.81 ± 0.03	−0.57 ± 0.03	19.6 ± 0.6	0.44 ± 0.02	−0.38 ± 0.02	4.68 ± 0.03	0.46 ± 0.002	−0.59 ± 0.002
Step 3	0.64 ± 0.01/ 0.76 ± 0.05a	−0.74 ± 0.005	−0.10 ± 0.004a	0.60 ± 0.004/ 0.68 ± 0.06 a	0.70 ± 0.001	− 0.05 ± 0.001a	0.36 ± 0.01	−0.23 ± 0.01	−0.44 ± 0.02
Step 4	–	–	–	–	–	–	0.12 ± 0.002	0.16 ± 0.01	0.95 ± 0.02

Apparent kinetic parameters derived from simultaneous fitting of normalized absorption and fluorescence traces into multi-exponential functions for reactions involving 3 µM M.HhaI (W41F/I86W), 2.5 µM DNA and 100 µM cofactor. Values are reported with standard errors of the fit. Steps 1–2 correspond to target base flipping and catalytic loop closure; step 3 - covalent bond formation; step 4 - covalent bond breakage, base flip-back and loop opening.

^aUnassigned change in fluorescence intensity.