Cooperative Binding of Midazolam with Testosterone and α-Naphthoflavone within the CYP3A4 Active Site: A NMR T1 Paramagnetic Relaxation Study

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Abstract
Recent studies have indicated that CYP3A4 exhibits non-Michaelis−Menten kinetics for numerous substrates. Both homo- and heterotropic activation have been reported, and kinetic models have suggested multiple substrates within the active site. We provide some of the first physicochemical data supporting the hypothesis of allosteric substrate binding within the CYP3A4 active site. Midazolam (MDZ) is metabolized by CYP3A4 to two hydroxylated metabolites, 1‘- and 4-hydroxymidazolam. Incubations using purified CYP3A4 and MDZ showed that both α-naphthoflavone (α-NF) and testosterone affect the ratio of formation rates of 1‘- and 4-hydroxymidazolam. Similar to previous reports, α-NF was found to promote formation of 1‘-hydroxymidazolam, while testosterone stimulated formation of 4-hydroxymidazolam. NMR was used to measure the closest approach of individual MDZ protons to the paramagnetic heme iron of CYP3A4 using paramagnetic T1 relaxation measurements. Solutions of 0.2 μM CYP3A4 with 500 μM MDZ resulted in calculated distances between 7.4 and 8.3 Å for all monitored MDZ protons. The distances were statistically equivalent for all protons except C3−H and were consistent with the rotation within the active site or sliding parallel to the heme plane. When 50 μM α-NF was added, proton−heme iron distances ranged from 7.3 to 10.0 Å. Consistent with kinetics of activation, the 1‘ position was situated closest to the heme, while the fluorophenyl 5-H proton was the furthest. Proton−heme iron distances for MDZ with CYP3A4 and 50 μM testosterone ranged from 7.7 to 9.0 Å, with the flourophenyl 5-H proton furthest from the heme iron and the C4-H closest to the heme, also consistent with kinetic observations. When titrated with CYP3A4 in the presence of MDZ, testosterone and α-NF resonances themselves exhibited significant broadening and enhanced relaxation rates, indicating that these effector molecules were also bound within the CYP3A4 active site near the paramagnetic heme iron. These results suggest that the effector exerts its cooperative effects on MDZ metabolism through simultaneous binding of MDZ and effector near the CYP3A4 heme.