Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes.

ABSTRACT: The natural catalytic cycle of cytochrome (cyt) P450 enzymes in human liver microsome (HLM) films was activated electrochemically via the electron transfer sequence electrode?cyt P450 reductase (CPR)?cyt P450. Cyclic voltammograms for HLM films had midpoint potentials of -0.50 V vs. SCE at pH 7.4 characteristic of CPR, not cyt P450s. HLM and CPR microsomes without cyt P450s did not electrocatalytically reduce H2O2, and did not shift midpoint potential when CO was added, also indicating that the peaks do not correspond to iron heme cyt P450 enzymes. Electrochemical activation of the natural cyt P450 cycle for substrate conversion via CPR in HLM films was confirmed by catalytic electrolysis in an electrochemical microfluidic array designed to generate and detect reactive metabolites by measuring their reactivity with DNA.

SUBMITTER: Wasalathanthri DP

PROVIDER: S-EPMC3636991 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes.

Wasalathanthri Dhanuka P DP Malla Spundana S Faria Ronaldo C RC Rusling James F JF

Electroanalysis 20121101 11

The natural catalytic cycle of cytochrome (cyt) P450 enzymes in human liver microsome (HLM) films was activated electrochemically via the electron transfer sequence electrode→cyt P450 reductase (CPR)→cyt P450. Cyclic voltammograms for HLM films had midpoint potentials of -0.50 V vs. SCE at pH 7.4 characteristic of CPR, not cyt P450s. HLM and CPR microsomes without cyt P450s did not electrocatalytically reduce H<sub>2</sub>O<sub>2</sub>, and did not shift midpoint potential when CO was added, als ...[more]

PMID: 23626453

Similar Datasets

Efficient bioelectronic actuation of the natural catalytic pathway of human metabolic cytochrome P450s.

Project description:Cytochrome (cyt) P450s comprise the enzyme superfamily responsible for human oxidative metabolism of a majority of drugs and xenobiotics. Electronic delivery of electrons to cyt P450s could be used to drive the natural catalytic cycle for fundamental investigations, stereo- and regioselective synthesis, and biosensors. We describe herein 30 nm nanometer-thick films on electrodes featuring excess human cyt P450s and cyt P450 reductase (CPR) microsomes that efficiently mimic the natural catalytic pathway for the first time. Redox potentials, electron-transfer rates, CO-binding, and substrate conversion rates confirmed that electrons are delivered from the electrode to CPR, which transfers them to cyt P450. The film system enabled electrochemical probing of the interaction between cyt P450 and CPR for the first time. Agreement of film voltammetry data with theoretical simulations supports a pathway featuring a key equilibrium redox reaction in the natural catalytic pathway between reduced CPR and cyt P450 occurring within a CPR-cyt P450 complex uniquely poised for substrate conversion.

| S-EPMC3033457 | biostudies-literature

Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes.

Project description:AimThe most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin.MethodsThe metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4'-hydroxylation (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1'-hydroxylation/testosterone 6?-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences.ResultsThe samples of the two ethnic groups were not significantly different in cytochrome-b(5) concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A.ConclusionsThese data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins.

| S-EPMC3269586 | biostudies-literature

Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes.

Project description:The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2'-hydroxy-, 4'-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2'-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities. Molecular docking analysis showed that the C2'-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2' and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

| S-EPMC6438780 | biostudies-literature

Diversification of catalytic function in a synthetic family of chimeric cytochrome p450s.

Project description:We report initial characterization of a synthetic family of more than 3000 cytochrome P450s made by SCHEMA recombination of 3 bacterial CYP102s. A total of 16 heme domains and their holoenzyme fusions with each of the 3 parental reductase domains were tested for activity on 11 different substrates. The results show that the chimeric enzymes have acquired significant functional diversity, including the ability to accept substrates not accepted by the parent enzymes. K-means clustering analysis of the activity data allowed the enzymes to be classified into five distinct groups based on substrate specificity. The substrates can also be grouped such that one can be a "surrogate" for others in the group. Fusion of a functional chimeric heme domain with a parental reductase domain always reconstituted a functional holoenzyme, indicating that key interdomain interactions are conserved upon reductase swapping.

| S-EPMC1991292 | biostudies-literature

Characterization of the metabolism of fenretinide by human liver microsomes, cytochrome P450 enzymes and UDP-glucuronosyltransferases.

Project description:Background and purposeFenretinide (4-HPR) is a retinoic acid analogue, currently used in clinical trials in oncology. Metabolism of 4-HPR is of particular interest due to production of the active metabolite 4'-oxo 4-HPR and the clinical challenge of obtaining consistent 4-HPR plasma concentrations in patients. Here, we assessed the enzymes involved in various 4-HPR metabolic pathways.Experimental approachEnzymes involved in 4-HPR metabolism were characterized using human liver microsomes (HLM), supersomes over-expressing individual human cytochrome P450s (CYPs), uridine 5'-diphospho-glucoronosyl transferases (UGTs) and CYP2C8 variants expressed in Escherichia coli. Samples were analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry assays and kinetic parameters for metabolite formation determined. Incubations were also carried out with inhibitors of CYPs and methylation enzymes.Key resultsHLM were found to predominantly produce 4'-oxo 4-HPR, with an additional polar metabolite, 4'-hydroxy 4-HPR (4'-OH 4-HPR), produced by individual CYPs. CYPs 2C8, 3A4 and 3A5 were found to metabolize 4-HPR, with metabolite formation prevented by inhibitors of CYP3A4 and CYP2C8. Differences in metabolism to 4'-OH 4-HPR were observed with 2C8 variants, CYP2C8*4 exhibited a significantly lower V(max) value compared with *1. Conversely, a significantly higher V(max) value for CYP2C8*4 versus *1 was observed in terms of 4'-oxo formation. In terms of 4-HPR glucuronidation, UGTs 1A1, 1A3 and 1A6 produced the 4-HPR glucuronide metabolite.Conclusions and implicationsThe enzymes involved in 4-HPR metabolism have been characterized. The CYP2C8 isoform was found to have a significant effect on oxidative metabolism and may be of clinical relevance.

| S-EPMC3042207 | biostudies-literature

Catalytic versus inhibitory promiscuity in cytochrome P450s: implications for evolution of new function.

Project description:Catalytically promiscuous enzymes are intermediates in the evolution of new function from an existing pool of protein scaffolds. However, promiscuity will only confer an evolutionary advantage if other useful properties are not compromised or if there is no "negative trade-off" induced by the mutations that yield promiscuity. Therefore, identification and characterization of negative trade-offs incurred during the emergence of promiscuity are required to further develop the evolutionary models and to optimize in vitro evolution. One potential negative trade-off of catalytic promiscuity is increased susceptibility to inhibition, or inhibitory promiscuity. Here we exploit cytochrome P450s (CYPs) as a model protein scaffold that spans a vast range of catalytic promiscuity and apply a quantitative index to determine the relationship between promiscuity of catalysis and promiscuity of inhibition for a series of homologues. The aim of these studies is to begin to identify properties that, in general, correlate with catalytic promiscuity, hypothetically such as inhibitory promiscuity. Interestingly, the data indicate that the potential negative trade-off of inhibitory promiscuity is nearly insignificant because even highly substrate specific CYPs have high inhibitory promiscuity, with little incremental increase in susceptibility to inhibitory interactions as the substrate promiscuity increases across the series of enzymes. In the context of evolution, inhibitory promiscuity is not an obligate negative trade-off for catalytic promiscuity.

| S-EPMC3068220 | biostudies-literature

Non-natural olefin cyclopropanation catalyzed by diverse cytochrome P450s and other hemoproteins.

Project description:Recent work has shown that engineered variants of cytochrome P450BM3 (CYP102A1) efficiently catalyze non-natural reactions, including carbene and nitrene transfer reactions. Given the broad substrate range of natural P450 enzymes, we set out to explore if this diversity could be leveraged to generate a broad panel of new catalysts for olefin cyclopropanation (i.e., carbene transfer). Here, we took a step towards this goal by characterizing the carbene transfer activities of four new wild-type P450s that have different native substrates. All four were active and exhibited a range of product selectivities in the model reaction: cyclopropanation of styrene by using ethyl diazoacetate (EDA). Previous work on P450BM3 demonstrated that mutation of the axial coordinating cysteine, universally conserved among P450 enzymes, to a serine residue, increased activity for this non-natural reaction. The equivalent mutation in the selected P450s was found to activate carbene transfer chemistry both in vitro and in vivo. Furthermore, serum albumins complexed with hemin were also found to be efficient in vitro cyclopropanation catalysts.

| S-EPMC4287214 | biostudies-literature

Human liver microsomes study on the inhibitory effect of plantainoside D on the activity of cytochrome P450 activity.

Project description:BackgroundPlantainoside D is widely existed in the herbs and possesses various pharmacological activities, making it possible to co-administrate with other herbs. Its effect on cytochrome P450 enzymes (P450) is a risk factor for inducing adverse drug-drug interactions. To assess the effect of plantainoside D on the activity of major P450 isoenzymes in human liver microsomes.MethodsThe Cocktail method was conducted in human liver microsomes in the presence of probe substrates. The activity of P450 isoenzymes was evaluated by the production of corresponding metabolites. The concentration-dependent and time-dependent inhibition assays were performed in the presence of 0, 2.5, 5, 10, 25, 50, and 100 μM plantainoside D to characterize the inhibitory effect of plantainoside D.ResultsSignificant inhibition was observed in the activity of CYP1A2, 2D6, and 3A, which was concentration-dependent with the IC50 values of 12.83, 8.39, and 14.66 μM, respectively. The non-competitive manner and competitive manner were observed in the CYP3A inhibition (Ki = 7.16 μM) and CYP1A2 (Ki = 6.26 μM) and 2D6 inhibition (Ki = 4.54 μM), respectively. Additionally, the inhibition of CYP3A was found to be time-dependent with the KI of 1.28 μM-1 and Kinact of 0.039 min-1.ConclusionsWeak inhibitory effects of plantainoside D on the activity of CYP1A2, 2D6, and 3A were revealed in vitro, implying its potential of inducing interactions with CYP1A2-, 2D6-, and 3A-metabolized drugs. Although further in vivo validations are needed, the feasibility of the Cocktail method in evaluating P450 activity has been verified.

| S-EPMC9308932 | biostudies-literature

Characterization of the metabolism of eupalinolide A and B by carboxylesterase and cytochrome P450 in human liver microsomes.

Project description:Eupalinolide A (EA; Z-configuration) and eupalinolide B (EB; E-configuration) are bioactive cis-trans isomers isolated from Eupatorii Lindleyani Herba that exert anti-inflammatory and antitumor effects. Although one pharmacokinetic study found that the metabolic parameters of the isomers were different in rats, metabolic processes relevant to EA and EB remain largely unknown. Our preliminary findings revealed that EA and EB are rapidly hydrolyzed by carboxylesterase. Here, we investigated the metabolic stability and enzyme kinetics of carboxylesterase-mediated hydrolysis and cytochrome P450 (CYP)-mediated oxidation of EA and EB in human liver microsomes (HLMs). We also explored differences in the hydrolytic stability of EA and EB in human liver microsomes and rat liver microsomes (RLMs). Moreover, cytochrome P450 reaction phenotyping of the isomers was performed via in silico methods (i.e., using a quantitative structure-activity relationship model and molecular docking) and confirmed using human recombinant enzymes. The total normalized rate approach was considered to assess the relative contributions of five major cytochrome P450s to EA and EB metabolism. We found that EA and EB were eliminated rapidly, mainly by carboxylesterase-mediated hydrolysis, as compared with cytochrome P450-mediated oxidation. An inter-species difference was observed as well, with faster rates of EA and EB hydrolysis in rat liver microsomes. Furthermore, our findings confirmed EA and EB were metabolized by multiple cytochrome P450s, among which CYP3A4 played a particularly important role.

| S-EPMC9905117 | biostudies-literature

Geobacter sulfurreducens cytochrome c peroxidases: electrochemical classification of catalytic mechanisms.

Project description:Bacterial cytochrome c peroxidase (CcP) enzymes are diheme redox proteins that reduce hydrogen peroxide to water. They are canonically characterized by a peroxidatic (called L, for "low reduction potential") active site heme and a secondary heme (H, for "high reduction potential") associated with electron transfer, and an enzymatic activity that exists only when the H-heme is prereduced to the Fe(II) oxidation state. The prereduction step results in a conformational change at the active site itself, where a histidine-bearing loop will adopt an "open" conformation allowing hydrogen peroxide to bind to the Fe(III) of the L-heme. Notably, the enzyme from Nitrosomonas europaea does not require prereduction. Previously, we have shown that protein film voltammetry (PFV) is a highly useful tool for distinguishing the electrocatalytic mechanisms of the Nitromonas type of enzyme from other CcPs. Here, we apply PFV to the recently described enzyme from Geobacter sulfurreducens and the Geobacter S134P/V135K double mutant, which have been shown to be similar to members of the canonical subclass of peroxidases and the Nitrosomonas subclass of enzymes, respectively. Here we find that the wild-type Geobacter CcP is indeed similar electrochemically to the bacterial CcPs that require reductive activation, yet the S134P/V135K mutant shows two phases of electrocatalysis: one that is low in potential, like that of the wild-type enzyme, and a second, higher-potential phase that has a potential dependent upon substrate binding and pH yet is at a potential that is very similar to that of the H-heme. These findings are interpreted in terms of a model in which rate-limiting intraprotein electron transfer governs the catalytic performance of the S134P/V135K enzyme.

| S-EPMC3724354 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data