ol. Generally, enzyme reactions were prewarmed for a few minutes at 37uC in the presence of 50 mg of individual CYP2A6 variant proteins and various concentration of 8-methoxypsoralen ranging from 05 mM, together with coumarin at the final concentration of 10 mM. NADPH generating system having the highest dock score has the most favourable interaction. This procedure was performed first on the wild type CYP2A6 and subsequently repeated for the mutants. For the virtual mutation of CYP2A6, the Build Mutant Protocol was used for substitution of amino acid residues. Four mutants were generated using 1Z11 as the template by substituting the residues in the protein sequence. Energy minimization for optimization of residue geometry was applied to the mutant proteins using the algorithm of smart minimization until the gradient tolerance was satisfied. 8-MOP was docked in the same binding site as in the case for the wild type. Results and Discussion Functional characterization of polymorphic CYP variants is imperative in defining the magnitude of genetic alterations in the enzymatic activity. To date, CYP2A6 is known as one of the highly polymorphic CYP enzymes in human. Our group previously constructed several mutant alleles of CYP2A6, namely CYP2A615, CYP2A616, CYP2A621 and CYP2A622, and we demonstrated that point mutations in CYP2A6 primary sequence have minimum impact on the catalytic functions except for CYP2A622, of which the metabolic activity was evidently reduced in relative to the wild type CYP2A61. Despite low frequency of this variant reported among the Caucasian population, these findings have at least indicated that individual carriers of the homologous CYP2A622 allele is predicted to have diminished coumarin hydroxylase activity. Even though our study showed that no purchase Varlitinib significant difference in intrinsic clearance for CYP2A615, CYP2A616 and CYP2A621 in coumarin 7-hydroxylation when compared to the wild type, there were however apparent differences in the Km values of these variants. The variable Km values observed for the variants indicate that there are subtle yet significant changes in the 
active site induced by the mutations present in their primary sequences. To gain more insights into the ligand binding and mechanism of structural and functional changes as a result of the mutations, we further explored the inhibitory effect of the typical CYP2A6 inhibitor, 8-MOP on the variants, and compared the inhibitory effect with the binding affinity for coumarin. The differential ligand binding affinity was further rationalized by virtual mutation of CYP2A6 and docking of 8-MOP into the various CYP2A6 molecular models we have generated. Bacterial membrane fractions expressing human CYP2A6 wild type and variant proteins were exposed to several concentration of 8-MOP ranging from 05 mM, according to the protocol described under `Materials PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19653627 and Methods’. The inhibitory effect of 8-MOP in coumarin 7-hydroxylation of wild-type and variant CYP2A615, CYP2A616, CYP2A621 and CYP2A622 are illustrated in Fig. 1 of which IC50 values were determined from the curve plots. The IC50 value of 8-MOP in coumarin metabolism of wild-type and variants were estimated to be 0.1960.05, 2.3960.03, 0.4360.10, 1.5660.21 and 1.0560.21 mM respectively. Each value was resolved from the means of three independent experiments. One-way ANOVA All values are shown as the means 6 SD of three independent determinations. The Km values were determined in our previous study and li