Enzymatic Kinetics Study On Human Cytochrome P450 3A4 Polymorphic Alleles And Hybrid 3A43/3A4

The human cytochrome P450(CYP) superfamily members metabolize a vast array of clinically, physiologically, and toxicologically important compounds. CYP3A4 is the most abundant isoform of CYP in adult human liver and is involved in the metabolism of 45-60% of the drugs metabolized by CYPs. The expression of CYP3A4 varies 40-fold in individual human livers, and metabolism of CYP3A4 substrates varies at least 10-fold in vivo. This variation can affect drug efficacy and toxicity. One of the causes for these drug metabolism variations is genetic polymorphism. To date, several CYP3A4 alleles have been found and may be a source of inter-individual drug metabolism difference.The recombinant plasmids of CYP3A4 prototype and alleles, hybrid CYP3A43/3A4 were constructed via overlap-extention PCR mutagenesis. Yeast strain was chosen for expression. The postmitochondrial supernant (S9) fractions containing microsomes were isolated and were used as an enzyme source for biochemical analysis. Galactose induction of these genes in yeast produced high levels of human CYP3A4 enzyme, averaging approximately 15-20 mg of microsome proteins per milli-liter of culture. The biochemical activity was measured using fluorogenic substrate DBF and BOMCC in real-time kinetic assays. The fluorogenic substrate produced robust activity which could be inhibited in a dose-dependent manner with a known CYP3A4 inhibitor ketoconazole. In real-time kinetic assays, enzyme- and substrate-concentration dependent catalytic activities were demonstrated for these enzymes. The basic reaction condition was optimized that DBF concentration was 1μmol/L or BOMCC was 10μmol/L as well as microsomes were 0.25 mg per milli-liter in standardized assays.These results show budding yeast S.cerevisiae is a suitable host to express human CYP3A4 enzymes. It has the obvious advantage in that it is easier to work with, cheaper to grow, genetics are well understood, and many expression vectors are available. In addition, many human CYP proteins have been expressed in yeast and have been validated with reference drug standards to display native biochemical properties. Taking all these into account, the yeast represents the most convenient method of cloning and expressing large number of CYP variants. Secondly, A Km value of approximately 1.0μmol/L is obtained with DBF, a value which is comparable to published data. Thirdly, the value of Vmax/Km indicates that the R162Q and V170I mutations affect metabolism of DBF and BOMCC in a similar fashion. The T185S,P218V,S222P and L293P mutations exhibit significantly lower than prototype and especially DBF kinetic analyses show about 2.7-fold difference in the Km values between the prototype and the L293P variant, while the G56D allele exhibits higher than prototype. On the other hand, the catalytic activity of CYP3A43/3A4 hybrid enzyme closely paralleles that of CYP3A4 prototype.The panel of CYP3A4 polymorphic enzymes will be tested against various drugs and kinds of fluorogenic substrates to identify potentially adverse drug-CYP in order to generate predictive information applicable in personalized medicine.