Diversity in the Oxidation of Substrates by Cytochrome P450 2D6: Lack of an Obligatory Role of Aspartate 301-Substrate Electrostatic Bonding
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- 作者：F. Peter Guengerich ; Grover P. Miller ; Imad H. Hanna ; Martha V. Martin ; Serge Lé ; ger ; Cameron Black ; Nathalie Chauret ; José ; M. Silva ; Laird A. Trimble ; James A. Yergey ; and Deborah A. Nic
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：September 10, 2002
- 年：2002
- 卷：41
- 期：36
- 页码：11025 - 11034
- 全文大小：159K
- 年卷期：v.41,no.36(September 10, 2002)
- ISSN：1520-4995
文摘
Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquinehydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basicnitrogen. Residue Asp301 has been characterized as being involved in electrostatic interactions withsubstrates on the basis of homology modeling and site-directed mutagenesis experiments [Ellis, S. W.,Hayhurst, G. P., Smith, G., Lightfoot, T., Wong, M. M. S., Simula, A. P., Ackland, M. J., Sternberg, M.J. E., Lennard, M. S., Tucker, G. T., and Wolf, C. R. (1995) J. Biol. Chem. 270, 29055-29058]. However,pharmacophore models based on the role of Asp301 in substrate binding are compromised by reports ofcatalytic activity toward substrates devoid of a basic nitrogen, which have generally been ignored. Wecharacterized a high-affinity ligand for P450 2D6, also devoid of a basic nitrogen atom, spirosulfonamide[4-[3-(4-fluorophenyl)-2-oxo-1-oxaspiro[4.4]non-3-en-4-yl]benzenesulfonamide], with Ks 1.6 
M. Spirosulfonamide is a substrate for P450 2D6 (kcat 6.5 min-1 for the formation of a syn spiromethylene carbinol,Km 7 M). Mutation of Asp301 to neutral residues (Asn, Ser, Gly) did not substantially affect the bindingof spirosulfonamide (Ks 2.5-3.5 M). However, the hydroxylation of spirosulfonamide was attenuatedin these mutants to the same extent (90%) as for the classic nitrogenous substrate bufuralol, and the effectof the D301N substitution was manifested on kcat but not Km. Analogues of spirosulfonamide were alsoevaluated as ligands and substrates. Analogues in which the sulfonamide moiety was modified to anamide, thioamide, methyl sulfone, or hydrogen were ligands with Ks values of 1.7-32 M. All weresubstrates, and the methyl sulfone analogue was oxidized to the syn spiromethylene carbinol analogue ofthe major spirosulfonamide product. The D301N mutation produced varying changes in the oxidationpatterns of the spirosulfonamide analogues. The peptidometic ritonavir and the steroids progesterone andtestosterone had been reported to be substrates for P450 2D6, but the affinities (Ks) were unknown; thesewere estimated to be 1.2, 1.5, and 15 M, respectively (cf. 6 M for the classic substrate bufuralol). Theresults are consistent with a role of Asp301 other than electrostatic interaction with a positively chargedligand. H-Bonding or electrostatic interactions probably enhance binding of some substrates, but our resultsshow that it is not required for all substrates and explain why predictive models fail to recognize theproclivity for many substrates, especially those containing no basic nitrogen.
M. Spirosulfonamide is a substrate for P450 2D6 (kcat 6.5 min-1 for the formation of a syn spiromethylene carbinol,Km 7 M). Mutation of Asp301 to neutral residues (Asn, Ser, Gly) did not substantially affect the bindingof spirosulfonamide (Ks 2.5-3.5 M). However, the hydroxylation of spirosulfonamide was attenuatedin these mutants to the same extent (90%) as for the classic nitrogenous substrate bufuralol, and the effectof the D301N substitution was manifested on kcat but not Km. Analogues of spirosulfonamide were alsoevaluated as ligands and substrates. Analogues in which the sulfonamide moiety was modified to anamide, thioamide, methyl sulfone, or hydrogen were ligands with Ks values of 1.7-32 M. All weresubstrates, and the methyl sulfone analogue was oxidized to the syn spiromethylene carbinol analogue ofthe major spirosulfonamide product. The D301N mutation produced varying changes in the oxidationpatterns of the spirosulfonamide analogues. The peptidometic ritonavir and the steroids progesterone andtestosterone had been reported to be substrates for P450 2D6, but the affinities (Ks) were unknown; thesewere estimated to be 1.2, 1.5, and 15 M, respectively (cf. 6 M for the classic substrate bufuralol). Theresults are consistent with a role of Asp301 other than electrostatic interaction with a positively chargedligand. H-Bonding or electrostatic interactions probably enhance binding of some substrates, but our resultsshow that it is not required for all substrates and explain why predictive models fail to recognize theproclivity for many substrates, especially those containing no basic nitrogen.