. Author manuscript; available in PMC: 2007 Sep 7.

Published in final edited form as: J Steroid Biochem Mol Biol. 2006 Aug 4;101(1):50–60. doi: 10.1016/j.jsbmb.2006.06.004

Table 3.

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

	3β-HSD¹			Isomerase NADH²
Purified Enzyme	K_m μM	k_cat min⁻¹	k_cat/K_m min⁻¹ μM⁻¹	K_m μM	k_cat min⁻¹	k_cat/K_m min⁻¹ μM⁻¹
3β-HSD_1	34.1	3.5	0.10	4.6	45	9.8
H232A	N.D.³	N.D.	N.D.	2.0	1.2	0.6
N323L	94.3	2.9	0.03	2.9	33	11
S322A	19.0	1.0	0.05	3.3	16	4.8
N100A	N.D.	N.D.	N.D.	N.S.⁴	N.S.	N.S.
N100S	N.D.	N.D.	N.D.	N.S.	N.S.	N.S.
E126L	16.8	1.2	0.07	N.S.	N.S	N.S.

Kinetic constants for the 3β-HSD cofactor were determined in incubations containing NAD⁺ (13–200 μM), dehydroepiandrosterone (DHEA, 100 μM) and purified enzyme (0.03 mg) in 0.02 M potassium phosphate, pH 7.4. The values represent the means values from duplicate assays with standard deviations ≤ 6%.

Kinetic constants for the isomerase cofactor were determined in incubations of NADH (2–50 μM), 5-androstene-3,17-dione (50 μM) and purified enzyme (0.035 mg) in 0.02 M potassium phosphate buffer, pH 7.4.

N.D., not determined because there was no detectable activity with DHEA substrate.

⁴

N.S., NADH did not stimulate isomerase above the low basal activity with substrate alone (2–3% of wild-type isomerase)