Table 3. Comparison of steady-state parameters obtained for the isomerization reaction of wild-type and H121A BCO with cholest-5-en-3-one as substrate in stopped-flow experiments.

Measurements were in 50 mM potassium phosphate buffer (pH 7.5), 1% propan-2-ol, 1% Thesit and 100 mM glucose (with the addition of 6 nM glucose oxidase and 0.7 μM catalase for the measurements starting from the reduced enzyme form), at 25 °C.

		kcat (s−1)	Km,chol-5-en-3-one (mM)
Wild-type	Oxidized	32.3±3.5	0.14±0.04
	Reduced	23.3±2.5	0.17±0.01
H121A	Oxidized	21.5±1.9	0.11±0.03
	Reduced	29.8±1.8	0.14±0.02