Substrate proton to heme distances in CYP2C9 allelic variants and alterations by the heterotropic activator, dapsone

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Abstract

CYP2C9 polymorphisms result in reduced enzyme catalytic activity and greater activation by effector molecules as compared to wild-type protein, with the mechanism(s) for these changes in activity not fully elucidated. Through T1 NMR and spectral binding analyses, mechanism(s) for these differences in behavior of the variant proteins (CYP2C9.2, CYP2C9.3, and CYP2C9.5) as compared to CYP2C9.1 were assessed. Neither altered binding affinity nor substrate (flurbiprofen) proton to heme-iron distances differed substantially among the four enzymes. Co-incubation with dapsone resulted in reduced substrate proton to heme-iron distances for all enzymes, providing at least a partial mechanism for the activation of CYP2C9 variants by dapsone. In summary, neither altered binding affinity nor substrate orientation appear to be major factors in the reduced catalytic activity noted in the CYP2C9 variants, but dapsone co-incubation caused similar changes in substrate proton to heme-iron distances suggesting at least partial common mechanisms in the activation of the CYP2C9 forms.

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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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    Present address: Mylan Laboratories, Morgantown, WV 26505, USA.

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