. Author manuscript; available in PMC: 2009 Jul 1.

Published in final edited form as: J Steroid Biochem Mol Biol. 2008 May 3;111(1-2):66–73. doi: 10.1016/j.jsbmb.2008.04.007

Table 3.

Cofactor kinetics for the 3β-HSD and isomerase activities of the purified mutant and wild-type enzymes.

	3β-HSD¹			Isomerase²
Purified Enzyme	K_m µM	k_cat min⁻¹	k_cat/K_m min⁻¹ uM⁻¹	K_m µM	k_cat min⁻¹	k_cat/K_m min⁻¹ uM⁻¹
M187T	15.3 ± 1.0	6.0 ± 0.4	0.32 ± 0.02	0.5 ± 0.03	90.8 ± 5.3	181 ± 0.07
S124T	37.4 ± 2.2	5.7 ± 0.3	0.15 ± 0.01	2.6 ± 0.2	86.1 ± 5.8	33 ± 0.09
3β-HSD1	34.1 ± 1.7	3.5 ± 0.2	0.10 ± 0.005	4.6 ± 0.2	45.0 ± 1.8	9.8 ± 0.4
3β-HSD2	86.3 ± 5.6	7.1 ± 0.6	0.08 ± 0.005	12.6 ± 0.9	99.1 ± 6.4	7.9 ± 0.5

Kinetic constants for the 3β-HSD cofactor were determined in incubations containing NAD⁺ (10–200 µM), dehydroepiandrosterone (100 µM) and purified enzyme (0.03 mg) in 0.02 M potassium phosphate, pH 7.4.

Kinetic constants for the isomerase cofactor were determined in incubations of NADH (0–50 µM), 5-androstene-3,17-dione (100 µM) and purified enzyme (0.02 mg) in 0.02 M potassium phosphate buffer, pH 7.4. All values are the means of triplicate determinations ± standard deviations.