Table 2. Kinetic analysis of four-way junction cleavages by SsoHjc and SsoHje mutants.

Enzyme	kcat (min−1) 35°C (× 103)	kcat (min−1) 65°C (× 103)	kcat mutant/kcat WT	KDb(nM)
Hjc WT	27 ± 1	240a	1	1
Hjc S32A	—	<0.5c	<2 × 10−3	1.2 ± 0.1
Hje WT	1000 ± 50	8800a	1	—
Hje S30A	—	5.7 ± 0.7	6 × 10−4	—
Hje S30C	—	2.8 ± 0.1	3 × 10−4	—
Hje S30T	—	13 ± 0.3	1.5 × 10−3	—

^aValues for k_cat for wild-type Hjc and Hje were measured at 35°C, as the rates were too fast at 65°C to allow accurate measurement. These values were estimated based on the observation that the activity of Hje doubles for each 10°C increase in the reaction temperature.

^bWild-type Hje does not retard labelled four-way DNA junctions at concentrations below 1 μM protein under electrophoretic gel retardation conditions.

^cValues of k_cat <0.5 × 10⁻³ min⁻¹ could not be measured. The rates for mutants where no activity could be detected are therefore recorded as ‘<0.5’ above. Actual rates under these conditions may be far <0.5 × 10⁻³ min⁻¹.