Six Novel NonsynonymousCYP1A2Gene Polymorphisms: Catalytic Activities of the Naturally Occurring Variant Enzymes

Abstract

Six novel nonsynonymous nucleotide alterations were found in the cytochrome P450 1A2 gene in a Japanese population, which resulted in the following amino acid substitutions: T83M, E168Q, F186L, S212C, G299A, and T438I. These individuals were heterozygous for the amino acid substitutions. The potential functional alterations caused by the amino acid substitutions were characterized by a cDNA-mediated expression system using Chinese hamster V79 cells. Among the six CYP1A2 variants, F186L showed the most profound and statistically significant reduction in O-deethylation of phenacetin and 7-ethoxyresorufin. Kinetic analyses performed for the ethoxyresorufin O-deethylation revealed that the V max of the F186L variant was approximately 5% of that of the CYP1A2 wild type, despite a 5-fold lower K m value of the variant, the consequence of which was reduced enzymatic activity toward the substrate. Thus, for the first time, phenylalanine at residue 186 is suggested to be a critical amino acid for catalytic activity.

FUNCTIONAL CHARACTERIZATION OF THREE NATURALLY OCCURRING SINGLE NUCLEOTIDE POLYMORPHISMS IN THECES2GENE ENCODING CARBOXYLESTERASE 2 (HCE-2)

“…The cells were grown to reach approximately 70% confluency and rinsed with serumfree OPTI-MEM (Invitrogen) before transfection. The pCR3.1, pCR3.1/CES2 wild-type, pCR3.1/CES2 R34W, and pCR3.1/CES2 V142M plasmids (6 g each) were transfected individually using the LipofectAMINE PLUS reagent (Invitrogen) as described previously (Murayama et al, 2004). The cells were harvested after 48 h and homogenized in 100 mM potassium phosphate buffer (pH 7.4).…”

Pharmacogenetics and pharmacogenomics of schizophrenia: a review of last decade of research

“…43,51 Several polymorphisms have been reported in the CYP1A2, with three variants showing decreased activity (*1C, *1K and *11). 67,68 In addition, smoking seriously affects the activity of CYP1A2, with smokers displaying a higher metabolic rate, 69 in particular those with *1C and *1D variants. 70 However, CYP1A2 polymorphisms did not significantly influence individuals' clozapine metabolic capacity, 71 although delays in response to clozapine have been observed in individuals with the UM phenotype.…”

Metabolism-Based Drug-Drug Interactions: What Determines Individual Variability in Cytochrome P450 Induction?

“…This finding suggests that this point mutation does not affect the constitutive expression of the gene but primarily influences the in vivo induction of CYP1A2 by smoking, presumably attributed to differential binding of a protein factor(s) to the wild-type and variant allele sequences (Nakajima et al, 1999). Kinetic analysis revealed that F186L, a CYP1A2 variant that was newly identified along with five other novel nonsynonymous nucleotide alterations in human CYP1A2 gene and displayed the most profound reduction in CYP1A2 activity, showed a V max only 5% of that of the CYP1A2 wild type, as measured for the ethoxyresorufin O-deethylation (Murayama et al, 2004). However, whether this variant is associated with altered inducibility of CYP1A2 remains unclear.…”