Substrate proton to heme distances in CYP2C9 allelic variants and alterations by the heterotropic activator, dapsone
Section snippets
Chemicals
D2O, polyvinylpyrrolidone, sodium dithionite, racemic-flurbiprofen, dapsone, and dilauroylphosphatidylcholine were purchased from Sigma–Aldrich (St. Louis, MO). Centricon MW cutoff filters were purchased from Millipore (Billerica, MA). Potassium phosphate, and EDTA were purchased from Fisher Scientific (Pittsburgh, PA). All other chemicals were purchased from commercial sources and were of the highest purity available.
Enzyme expression and purification
CYP2C9.1 and CYP2C9.3 were expressed in E. Coli according to established
Results
The chemical structures along with the proton numbering schemes used throughout for flurbiprofen and dapsone appear in Fig. 1.
Discussion
It is becoming evident that not only are drug–drug interactions involving P450s dependent on substrate and effector but also can be allelic variant dependent. For example, genetic variant dependent activation of CYP2C9 by dapsone has been demonstrated, in vitro[9]. In each variant, the catalytic efficiency was increased due to both a reduction in Km as well as an increase in Vmbut to varying degrees, depending on the protein. In these previous studies, the efficiency (Vm/Km) of flurbiprofen
Acknowledgments
This work was funded by NIH Grants #GM063215 and GM069753 to TST. The authors also acknowledge the assistance of Dr. Chuck Locuson and Dr. Murali Subramanian in conducting the spin state determinations.
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