Table 2. Specificity constants and free energy differences in ground state and transition state energies between wild-type and mutant 3α-HSD/CRs^a.

	WT	P185A	P185G	T188A	T188S
kcat/KiaKb (mM−2s−1)	42 ×104	8.3×104	0.40×104	1.7×104	1.4×104
ΔΔG‡ mut/wt (kcal/mol)		0.96	2.8	1.9	2.0
ΔΔGbmut/wt (kcal/mol)		1.7	2.6	1.8	1.9

^aThe differential binding energy for the ground-state enzyme-NAD⁺ complex and the transition state of the the reaction catalyzed by the wild-type and mutant 3α-HSD/CRs at pH 10.5 were calculated using Equation 4 and 5, respectively. ΔΔG_{b mut/wt} and ΔΔG ^‡ _mut/wt are the differential ground- and transition-state binding energies, respectively. k_cat/K_iaK_b is referred to k_cat/K_iNADK_androsterone obtained from Table 1.