Cooperative Binding of Midazolam with Testosterone and -Naphthoflavone within the CYP3A4 Active Site: A NMR T1 Par
详细信息 查看全文
- 作者：Michael D. Cameron ; Bo Wen ; Kyle E. Allen ; Arthur G. Roberts ; Jason T. Schuman ; A. Patricia Campbell ; Kent L. Kunze ; and Sidney D. Nelson
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：November 1, 2005
- 年：2005
- 卷：44
- 期：43
- 页码：14143 - 14151
- 全文大小：157K
- 年卷期：v.44,no.43(November 1, 2005)
- ISSN：1520-4995
文摘
Recent studies have indicated that CYP3A4 exhibits non-Michaelis-Menten kinetics fornumerous substrates. Both homo- and heterotropic activation have been reported, and kinetic modelshave suggested multiple substrates within the active site. We provide some of the first physicochemicaldata 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 
lpha.gif" BORDER=0>-naphthoflavone (lpha.gif" BORDER=0>-NF) and testosteroneaffect the ratio of formation rates of 1'- and 4-hydroxymidazolam. Similar to previous reports, lpha.gif" BORDER=0>-NF wasfound to promote formation of 1'-hydroxymidazolam, while testosterone stimulated formation of4-hydroxymidazolam. NMR was used to measure the closest approach of individual MDZ protons to theparamagnetic heme iron of CYP3A4 using paramagnetic T1 relaxation measurements. Solutions of 0.2
M CYP3A4 with 500 M MDZ resulted in calculated distances between 7.4 and 8.3 Å for all monitoredMDZ protons. The distances were statistically equivalent for all protons except C3-H and were consistentwith the rotation within the active site or sliding parallel to the heme plane. When 50 M lpha.gif" BORDER=0>-NF wasadded, proton-heme iron distances ranged from 7.3 to 10.0 Å. Consistent with kinetics of activation, the1' 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 M testosterone ranged from 7.7 to 9.0 Å, with theflourophenyl 5-H proton furthest from the heme iron and the C4-H closest to the heme, also consistentwith kinetic observations. When titrated with CYP3A4 in the presence of MDZ, testosterone and lpha.gif" BORDER=0>-NFresonances themselves exhibited significant broadening and enhanced relaxation rates, indicating that theseeffector 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 throughsimultaneous binding of MDZ and effector near the CYP3A4 heme.
lpha.gif" BORDER=0>-naphthoflavone (lpha.gif" BORDER=0>-NF) and testosteroneaffect the ratio of formation rates of 1'- and 4-hydroxymidazolam. Similar to previous reports, lpha.gif" BORDER=0>-NF wasfound to promote formation of 1'-hydroxymidazolam, while testosterone stimulated formation of4-hydroxymidazolam. NMR was used to measure the closest approach of individual MDZ protons to theparamagnetic heme iron of CYP3A4 using paramagnetic T1 relaxation measurements. Solutions of 0.2M CYP3A4 with 500 M MDZ resulted in calculated distances between 7.4 and 8.3 Å for all monitoredMDZ protons. The distances were statistically equivalent for all protons except C3-H and were consistentwith the rotation within the active site or sliding parallel to the heme plane. When 50 M lpha.gif" BORDER=0>-NF wasadded, proton-heme iron distances ranged from 7.3 to 10.0 Å. Consistent with kinetics of activation, the1' 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 M testosterone ranged from 7.7 to 9.0 Å, with theflourophenyl 5-H proton furthest from the heme iron and the C4-H closest to the heme, also consistentwith kinetic observations. When titrated with CYP3A4 in the presence of MDZ, testosterone and lpha.gif" BORDER=0>-NFresonances themselves exhibited significant broadening and enhanced relaxation rates, indicating that theseeffector 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 throughsimultaneous binding of MDZ and effector near the CYP3A4 heme.