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. Ile26...

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Veröffentlicht in:DRUG METABOLISM AND PHARMACOKINETICS 2008-01, Vol.23 (3), p.165-174
Hauptverfasser: Singh, Rajinder, Ting, Jonathan G., Pan, Yan, Teh, Lay Kek, Ismail, Rusli, Ong, Chin Eng
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container_issue 3
container_start_page 165
container_title DRUG METABOLISM AND PHARMACOKINETICS
container_volume 23
creator Singh, Rajinder
Ting, Jonathan G.
Pan, Yan
Teh, Lay Kek
Ismail, Rusli
Ong, Chin Eng
description 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.
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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.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>18574320</pmid><doi>10.2133/dmpk.23.165</doi><tpages>10</tpages></addata></record>
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ispartof DRUG METABOLISM AND PHARMACOKINETICS, 2008-01, Vol.23 (3), p.165-174
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source J-STAGE Free; MEDLINE; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry
subjects Amino Acid Substitution
Animals
Aryl Hydrocarbon Hydroxylases - genetics
Aryl Hydrocarbon Hydroxylases - metabolism
Catalysis
Catalytic Domain
Cloning, Molecular
CYP2C84
Cytochrome P-450 CYP2C8
DNA Primers
Endopeptidase K - metabolism
Enzyme Stability
Escherichia coli - genetics
genetic polymorphism
Heme - metabolism
Humans
Isoleucine - genetics
Isoleucine - physiology
Mutagenesis, Site-Directed
NADPH-Ferrihemoprotein Reductase - genetics
paclitaxel
Paclitaxel - metabolism
Paclitaxel - pharmacology
pharmacogenomics
Polymorphism, Genetic
Protein Folding
Rats
site-directed mutagenesis
Substrate Specificity
Tolbutamide - metabolism
Tolbutamide - pharmacology
title Functional Role of Ile264 in CYP2C8: Mutations Affect Haem Incorporation and Catalytic Activity
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