Functional role of Ile264 in CYP2C8: mutations affect haem incorporation and catalytic activity.
The work described in this study aimed to express CYP2C8 wild-type and mutant proteins in bacterial expression system and to use the expressed proteins to investigate the structural and functional consequences of a reported allele CYP2C8(*)4 (carrying Ile264Met substitution) on protein activity. Ile264 was replaced by three different amino acids resulting in three mutant constructs, 2C8I264M, 2C8I264R and 2C8I264D. The presence of isoleucine at position 264 in CYP2C8 was found to be important for proper haem insertion and protein folding; whereas bulkier or charged residues were highly disruptive resulting in inactive proteins with minimum spectral and catalytic activities. This was evidenced from the low levels of Soret peak at 450 nm and negligible levels of tolbutamide methylhydroxylase activity. Kinetic study using paclitaxel indicated that all three mutants exhibited only 9.7 to 35.4% of the activity level observed in the wild-type. In addition, the mutants were more sensitive to proteinase K digestion, indicating a possible alteration of conformation. The combined effects of protein instability and compromised catalytic activity resulted in defective CYP2C8 protein which may have clinical implications in carriers of CYP2C8*4, particularly in terms of their capacity to clear potent drugs and their susceptibility to adverse drug reactions.
Singh
Rajinder
R
School of Pharmacy and Health Sciences, International Medical University, Kuala Lumpur, Malaysia.
Ting
Jonathan G
JG
Pan
Yan
Y
Teh
Lay Kek
LK
Ismail
Rusli
R
Ong
Chin Eng
CE
D019943:Amino Acid Substitution; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D065727:Cytochrome P-450 CYP2C8; D017931:DNA Primers; D019286:Endopeptidase K / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007532:Isoleucine / Q000235:genetics* / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics; D017239:Paclitaxel / Q000378:metabolism / Q000494:pharmacology; D011110:Polymorphism, Genetic; D017510:Protein Folding; D051381:Rats; D013379:Substrate Specificity; D014044:Tolbutamide / Q000378:metabolism / Q000494:pharmacology
D017931:DNA Primers; D007532:Isoleucine; D006418:Heme; D014044:Tolbutamide; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D009251:NADPH-Ferrihemoprotein Reductase; D019286:Endopeptidase K; D017239:Paclitaxel