Diversity in the oxidation of substrates by cytochrome P450 2D6: lack of an obligatory role of aspartate 301-substrate electrostatic bonding.
Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Residue Asp301 has been characterized as being involved in electrostatic interactions with substrates 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 of catalytic activity toward substrates devoid of a basic nitrogen, which have generally been ignored. We characterized 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 K(s) 1.6 microM. Spirosulfonamide is a substrate for P450 2D6 (k(cat) 6.5 min(-)(1) for the formation of a syn spiromethylene carbinol, K(m) 7 microM). Mutation of Asp301 to neutral residues (Asn, Ser, Gly) did not substantially affect the binding of spirosulfonamide (K(s) 2.5-3.5 microM). However, the hydroxylation of spirosulfonamide was attenuated in these mutants to the same extent (90%) as for the classic nitrogenous substrate bufuralol, and the effect of the D301N substitution was manifested on k(cat) but not K(m). Analogues of spirosulfonamide were also evaluated as ligands and substrates. Analogues in which the sulfonamide moiety was modified to an amide, thioamide, methyl sulfone, or hydrogen were ligands with K(s) values of 1.7-32 microM. All were substrates, and the methyl sulfone analogue was oxidized to the syn spiromethylene carbinol analogue of the major spirosulfonamide product. The D301N mutation produced varying changes in the oxidation patterns of the spirosulfonamide analogues. The peptidometic ritonavir and the steroids progesterone and testosterone had been reported to be substrates for P450 2D6, but the affinities (K(s)) were unknown; these were estimated to be 1.2, 1.5, and 15 microM, respectively (cf. 6 microM for the classic substrate bufuralol). The results are consistent with a role of Asp301 other than electrostatic interaction with a positively charged ligand. H-Bonding or electrostatic interactions probably enhance binding of some substrates, but our results show that it is not required for all substrates and explain why predictive models fail to recognize the proclivity for many substrates, especially those containing no basic nitrogen.
Guengerich
F Peter
FP
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA. guengerich@toxicology.mc.vanderbuilt.edu
Miller
Grover P
GP
Hanna
Imad H
IH
Martin
Martha V
MV
Léger
Serge
S
Black
Cameron
C
Chauret
Nathalie
N
Silva
José M
JM
Trimble
Laird A
LA
Yergey
James A
JA
Nicoll-Griffith
Deborah A
DA
D000588:Amines / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D006860:Hydrogen Bonding; D008024:Ligands; D008862:Microsomes, Liver / Q000201:enzymology; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013141:Spiro Compounds / Q000737:chemistry*; D055672:Static Electricity; D013379:Substrate Specificity; D013449:Sulfonamides / Q000737:chemistry*; D000096926:Benzenesulfonamides
D000588:Amines; D001224:Aspartic Acid; D019389:Cytochrome P-450 CYP2D6; D008024:Ligands; D011994:Recombinant Proteins; D013141:Spiro Compounds; D013449:Sulfonamides; D000096926:Benzenesulfonamides; C465867:spirosulfonamide