TABLE 2.

Effects of Ala-scanning mutagenesis of Thr²⁶⁴, Ser⁴²⁰, and/or Ser⁴⁷⁸ on CYP3A4 structure and function

For experimental details see under “Experimental Procedures.” Values are mean ± S.D. of three separate determinations. Testosterone 6β-hydroxylase activity was determined with purified recombinant CYP3A4wt and mutant CYP3A4 proteins, functionally reconstituted with P450 reductase and cytochrome b₅ as described (50).

CYP3A4	Total CYP3A4 protein content	Holo-P450 content	Holo-P450/total P450 protein content	Testosterone 6β-hydroxylase activity
	nmol/ml	nmol/ml	nmol/ml	pmol 6β-hydroxytestosterone formed/pmol P450/min
Wild type	4.17 ± 0.15	3.49 ± 0.15	0.81 ± 0.17	15.0 ± 0.6
T264A	5.98 ± 0.35	4.25 ± 0.44	0.71 ± 0.21	11.9 ± 0.3
S420A	10.5 ± 0.75	8.71 ± 0.57	0.84 ± 0.21	15.6 ± 0.4
S478A	7.22 ± 0.65	5.38 ± 0.40	0.75 ± 0.17	14.4 ± 0.6
T264A/S420A	5.57 ± 0.20	4.54 ± 0.26	0.82 ± 0.15	12.5 ± 0.4
T264A/S478A	14.7 ± 0.38	11.1 ± 0.36	0.76 ± 0.13	12.6 ± 0.3
S420A/S478A	3.46 ± 0.15	2.53 ± 0.29	0.73 ± 0.19	14.2 ± 0.4
T264A/S420A/S478A	5.63 ± 0.30	4.34 ± 0.11	0.77 ± 0.13	17.5 ± 0.7