Publication: Differential contribution of active site residues in substrate recognition sites 1 and 5 to cytochrome P450 2C8 substrate selectivity and regioselectivity
1
Issued Date
2004-06-22
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00062960
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2-s2.0-2942711686
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Mahidol University
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SCOPUS
Bibliographic Citation
Biochemistry. Vol.43, No.24 (2004), 7834-7842
Suggested Citation
Oranun Kerdpin, David J. Elliot, Sanford L. Boye, Donald J. Birkett, Krongtong Yoovathaworn, John O. Miners Differential contribution of active site residues in substrate recognition sites 1 and 5 to cytochrome P450 2C8 substrate selectivity and regioselectivity. Biochemistry. Vol.43, No.24 (2004), 7834-7842. doi:10.1021/bi0496844 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/21178
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Title
Differential contribution of active site residues in substrate recognition sites 1 and 5 to cytochrome P450 2C8 substrate selectivity and regioselectivity
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Abstract
Selected active site residues in substrate recognition sites (SRS) 1 and 5 of cytochrome P450 2C8 (CYP2C8) were mutated to the corresponding amino acids present in CYP2C9 to investigate the contribution of these positions to the unique substrate selectivity and regioselectivity of CYP2C8. The effects of mutations, singly and in combination, were assessed from changes in the kinetics of paclitaxel 6α-hydroxylation, a CYP2C8-specific pathway, and the tolylmethyl and ring hydroxylations of torsemide, a mixed CYP2C9/CYP2C8 substrate. Within SRS 1, the single mutation S114F abolished paclitaxel 6α-hydroxylation, while the I113V substitution resulted in modest parallel reductions in Kmand Vmax. Mutations in SRS5 (viz., V362L, G365S, and V366L) reduced paclitaxel intrinsic clearance (Vmax/Km) by 88-100%. Torsemide is preferentially metabolized by CYP2C9, and it was anticipated that the mutations in CYP2C8 might increase activity. However, methyl and ring hydroxylation intrinsic clearances were either unchanged or decreased by the mutations, although hydroxylation regioselectivity was often altered relative to wild-type CYP2C8. The mutations significantly increased (28-968%) Kmvalues for both torsemide methyl and ring hydroxylation but had variable effects on Vmax. The effects of the combined mutations in SRS1, SRS5, and SRS1 plus SRS5 were generally consistent with the changes produced by the separate mutations. Mutation of CYP2C8 at position 359 (S359I), a site of genetic polymorphism in CYP2C9, resulted in relatively minor changes in paclitaxel- and torsemide-hydroxylase activities. The results are consistent with multiple substrate binding orientations within the CYP2C8 active site and a differential contribution of active site residues to paclitaxel and torsemide binding and turnover.