Journal of intravenous nursing

Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC50 values. In general, a similar trend of change in the IC50 and Km values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6 16, differences in IC50 values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6 16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins

Geometric changes in CYP2A6 wild type and mutant proteins.

<p><b>a)</b> Changes in the geometry of the protein tertiary structures. Wild type, red; CYP2A6*15, green; CYP2A6*16, blue; CYP2A6*21, black; CYP2A6*22, magenta; <b>b)</b> Superimposition of the wild type and mutant structures. Yellow circle indicates the binding site.</p

Velocity rates for coumarin 7-hydroxylation in incubations of coumarin (10 µM) without (open bars) or with (hatched bars) pre-incubation with 8-methoxypsoralen at 1.0 µM in CYP2A6 proteins.

<p>Data are shown as mean ± SD from triplicate determinations. Degree of statistical differences between incubations without and with pre-incubation as determined by two-tailed Student's <i>t</i> test: the mean difference is significant at 0.05 level (* p<0.05) and highly significant at 0.01 level (** p<0.01).</p

Binding of 8-methoxypsoralen in CYP2A6 mutant active sites.

<p>Side chains of amino acid residues within 5 Å of the ligand are rendered as stick figures. Red, oxygen atoms; blue, nitrogen atoms; gray, methoxsalen-complex carbon atoms; and magenta, heme group. a: CYP2A6*15; b: CYP2A6*16; c: CYP2A6*21; and d: CYP2A6*22.</p

The ribbon diagram showing the overall structure of the wild-type CYP2A6 with locations of mutated residues highlighted.

<p>Secondary structures α-helices and β-sheets are also shown and labelled. The heme moiety is indicated in sticks mode (magenta color).</p

Binding of 8-methoxypsoralen in CYP2A6 wild type active site.

<p>Side chains of amino acid residues within 5 Å of the ligand are rendered as stick figures. Red, oxygen atoms; blue, nitrogen atoms; gray, methoxsalen-complex carbon atoms; and magenta, heme group.</p

Representative IC₅₀ plots demonstrating the inhibitory effect of 8-methoxypsoralen at concentration of 0–5 µM on CYP2A6*1 (wild type) (•), CYP2A6*15 (▴), CYP2A6*16 (▪), CYP2A6*21 (♦) and CYP2A6*22 .

<p>Points are experimentally determined values while solid lines are the computer-generated curves of best fit (by SigmaPlot® programme). Data are presented as mean values from triplicate measurements.</p

Conformational changes of the CYP2A6 active site cavities.

<p><b>a)</b> Space-filling representation of amino acid residues forming the CYP2A6 active sites. a1: wild type; a2: CYP2A6*15; a3: CYP2A6*16; a4: CYP2A6*21; and a5: CYP2A6*22. <b>b)</b> The conformations of active site cavities depicted as stick models. b1: wild type; b2: CYP2A6*15; b3: CYP2A6*16; b4: CYP2A6*21; and b5: CYP2A6*22. The heme moiety is presented at the floor of active sites as magenta sticks.</p