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Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9.

ABSTRACT: The cytochrome P450 enzymes represent an important class of heme-containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological-based chips for standardizing cytochrome P450 metabolism or for high-throughput screening of pharmaceutical agents. Previous studies have generally immobilized P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely, superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed.

SUBMITTER: Gannett PM

PROVIDER: S-EPMC2556980 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9.

Gannett Peter M PM Kabulski Jarod J Perez Felio A FA Liu Zhongyuan Z Lederman David D Locuson Charles W CW Ayscue Robyn R RR Thomsen Nissa M NM Tracy Timothy S TS

Journal of the American Chemical Society 20060701 26

The cytochrome P450 enzymes represent an important class of heme-containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological-based chips for standardizing cytochrome P450 metabolism or for high-throughput screening of pharmaceutical agents. Previous studies have generally immobil ...[more]

PMID: 16802783

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Project description:Cytochrome P450 enzymes (P450s) are important in drug metabolism and have been linked to adverse drug reactions. P450s display broad substrate reactivity, and prediction of metabolites is complex. QM/MM studies of P450 reactivity have provided insight into important details of the reaction mechanisms and have the potential to make predictions of metabolite formation. Here we present a comprehensive study of the oxidation of three widely used pharmaceutical compounds (S-ibuprofen, diclofenac, and S-warfarin) by one of the major drug-metabolizing P450 isoforms, CYP2C9. The reaction barriers to substrate oxidation by the iron-oxo species (Compound I) have been calculated at the B3LYP-D/CHARMM27 level for different possible metabolism sites for each drug, on multiple pathways. In the cases of ibuprofen and warfarin, the process with the lowest activation energy is consistent with the experimentally preferred metabolite. For diclofenac, the pathway leading to the experimentally observed metabolite is not the one with the lowest activation energy. This apparent inconsistency with experiment might be explained by the two very different binding modes involved in oxidation at the two competing positions. The carboxylate of diclofenac interacts strongly with the CYP2C9 Arg108 side chain in the transition state for formation of the observed metabolite-but not in that for the competing pathway. We compare reaction barriers calculated both in the presence and in the absence of the protein and observe a marked improvement in selectivity prediction ability upon inclusion of the protein for all of the substrates studied. The barriers calculated with the protein are generally higher than those calculated in the gas phase. This suggests that active-site residues surrounding the substrate play an important role in controlling selectivity in CYP2C9. The results show that inclusion of sampling (particularly) and dispersion effects is important in making accurate predictions of drug metabolism selectivity of P450s using QM/MM methods.

Dataset Information

Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9.

Publications

Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9.

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