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1.
J Biosci Bioeng ; 116(4): 433-7, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23643620

ABSTRACT

We site-specifically conjugated biotin-PEG derivatives with spacer arms of different lengths to mutant P450cam (3mD) and evaluated the activity of and structural changes in the conjugates as a first step toward clarifying the mechanism whereby the activity of the 3mD conjugate is inhibited. 3mD was prepared by site-specific mutation to inhibit its enzymatic activity artificially, after which the derivative compounds were conjugated to the enzyme. 3mD has one cysteine on its surface with a reactive thiol group that can react with compounds near the active site, where a conformational change will be induced after conjugation. The activity of 3mD was retained in the biotin-PEG2-3mD conjugate, but was dramatically reduced in the biotin-PEG11-3mD conjugate. To investigate the effect of poly(ethylene glycol) (PEG) length on the enzymatic activity after conjugation, PEGs of different lengths, exceeding that in biotin-PEG11, and whose termini were not biotin, were conjugated to 3mD. The activity of 3mD decreased in all these conjugates. This indicates that the activity of 3mD in these conjugates decreased after its conjugation with PEG molecules that exceeded a certain length. The biotin-PEG2-3mD, which retains enzymatic activity after conjugation, showed avidin responsiveness; the enzymatic activity decreased after avidin binding.


Subject(s)
Avidin/metabolism , Biotin/metabolism , Camphor 5-Monooxygenase/chemistry , Camphor 5-Monooxygenase/metabolism , Avidin/chemistry , Biotin/chemistry , Camphor 5-Monooxygenase/antagonists & inhibitors , Camphor 5-Monooxygenase/genetics , Catalytic Domain , Cysteine/metabolism , Mutagenesis, Site-Directed , Polyethylene Glycols/chemistry
2.
J Biosci Bioeng ; 115(6): 639-44, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23369276

ABSTRACT

We conjugated a molecular recognition moiety, biotin, with an enzyme site-specifically near to its active site and succeeded in inactivating the enzyme by binding the specific target biomolecule avidin to biotin. Bacterial P450 was used as a model enzyme, which has attracted much attention in several fields. Site-directed mutagenesis was conducted to produce a mutant P450 that could attach biotin site-specifically. The activity of the conjugate decreased markedly to one tenth of that of biotinylated P450 after binding to avidin. Ultraviolet-visible spectroscopy of the carbon monoxide-bound P450, circular dichroism data, and the ratio of the active form to the sum of the active form and the inactive form indicated that this decrease in activity was because of a conformational change in the tertiary structure surrounding the active center after avidin binding, while the secondary structure of P450 remained unchanged.


Subject(s)
Biotin/chemistry , Camphor 5-Monooxygenase/chemistry , Avidin/chemistry , Avidin/metabolism , Biotin/metabolism , Camphor 5-Monooxygenase/antagonists & inhibitors , Camphor 5-Monooxygenase/genetics , Camphor 5-Monooxygenase/metabolism , Catalytic Domain , Mutagenesis, Site-Directed , Protein Conformation
3.
Anal Chem ; 81(2): 716-24, 2009 Jan 15.
Article in English | MEDLINE | ID: mdl-19099359

ABSTRACT

Studies of metabolic enzyme inhibition are necessary in drug development and toxicity investigations as potential tools to limit or prevent appearance of deleterious metabolites formed, for example, by cytochrome (cyt) P450 enzymes. In this paper, we evaluate the use of enzyme/DNA toxicity biosensors as tools to investigate enzyme inhibition. We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on pyrolytic graphite (PG) electrodes monitored via Ru(bpy)(3)(2+)-mediated DNA oxidation. Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the inhibitors, imidazole, imidazole-4-acetic acid, and sulconazole (in micromolar range) to monitor DNA damage inhibition. The initial rates of DNA damage decreased with increased inhibitor concentrations. Linear and nonlinear fits of Michaelis-Menten inhibition models were used to determine apparent inhibition constants (K(I)*) for the inhibitors. Elucidation of the best fitting inhibition model was achieved by comparing correlation coefficients and the sum of the square of the errors (SSE) from each inhibition model. Results confirmed the utility of the enzyme/DNA biosensor for metabolic inhibition studies. A simple competitive inhibition model best approximated the data for imidazole, imidazole-4-acetic acid and sulconazole with K(I)* of 268.2, 142.3, and 204.2 microM, respectively.


Subject(s)
Biosensing Techniques , Camphor 5-Monooxygenase/metabolism , DNA/chemistry , Enzyme Inhibitors/chemistry , 2,2'-Dipyridyl/analogs & derivatives , 2,2'-Dipyridyl/chemical synthesis , 2,2'-Dipyridyl/chemistry , Camphor 5-Monooxygenase/antagonists & inhibitors , Camphor 5-Monooxygenase/chemistry , DNA Damage/drug effects , Electrochemistry , Electrodes , Imidazoles/chemistry , Imidazoles/pharmacology , Kinetics , Nanotechnology , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistry , Potentiometry , Styrene/chemistry , Styrene/toxicity
4.
J Inorg Biochem ; 102(5-6): 1312-21, 2008.
Article in English | MEDLINE | ID: mdl-18331760

ABSTRACT

The effects of the divalent alkaline-earth metal ions (Ca2+ and Mg2+) on the substrate binding affinity, spin-state transition at the heme active site, conformational properties as well as the stability of the active form of cytochrome P450cam (CYP 101) have been investigated using various spectroscopic and kinetic methods. The divalent cations were found to have two types of effects on the enzyme. At the initial stage the alkaline-earth metal ion facilitated enhanced binding of the substrate and formation of the high-spin form of the heme active center of the enzyme compared to that in absence of any metal ion. However, analogous to many other mono-valent metal ions, the alkaline-earth metal ions were also less efficient than K+ in promoting the substrate binding and spin-transition properties of the enzyme. The auxiliary metal ions were shown to cause small but distinct change in the circular dichroism spectra of the substrate-free enzyme in the visible region, indicating that the tertiary structure around the heme was perturbed on binding of the auxiliary metal ion to the enzyme. The effect of the auxiliary metal ion was found to be more prominent in the WT enzyme compared to the Y96F mutant of P450cam suggesting that the Tyr 96 residue plays an important role in mediating the effects of the auxiliary metal ions to the active site of the enzyme. At the second stage of interaction, the alkaline-earth metal ions were found to slowly convert the enzyme into an inactive P420 form, which could be reversibly re-activated by addition of KCl. The results have been discussed in the light of understanding the mechanism of inactivation of certain mammalian P450 enzymes by these alkaline-earth metal ions.


Subject(s)
Calcium/pharmacology , Camphor 5-Monooxygenase/antagonists & inhibitors , Cations, Divalent/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Magnesium/pharmacology , Camphor/pharmacology , Camphor 5-Monooxygenase/chemistry , Circular Dichroism , Potassium Chloride/pharmacology , Spectrophotometry, Ultraviolet
5.
Biochem Biophys Res Commun ; 314(3): 810-6, 2004 Feb 13.
Article in English | MEDLINE | ID: mdl-14741708

ABSTRACT

The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis.


Subject(s)
Camphor 5-Monooxygenase/chemistry , Electrochemistry/methods , Spectrophotometry, Ultraviolet/methods , Camphor/chemistry , Camphor/metabolism , Camphor 5-Monooxygenase/antagonists & inhibitors , Camphor 5-Monooxygenase/metabolism , Carbon Monoxide/chemistry , Carbon Monoxide/pharmacology , Disulfides/chemistry , Dithionite/chemistry , Electrochemistry/instrumentation , Electrodes , Enzyme Inhibitors/pharmacology , Escherichia coli/metabolism , Gold/chemistry , Metyrapone/pharmacology , Oxidation-Reduction , Pseudomonas putida/enzymology , Pyridines/chemistry , Spectrophotometry, Ultraviolet/instrumentation
6.
J Am Chem Soc ; 124(35): 10254-5, 2002 Sep 04.
Article in English | MEDLINE | ID: mdl-12197708

ABSTRACT

We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Förster energy transfer upon binding (Kd = 0.83 muM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd approximately 0.02 muM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 A resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B', F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.


Subject(s)
Adamantane/analogs & derivatives , Camphor 5-Monooxygenase/antagonists & inhibitors , Camphor 5-Monooxygenase/chemistry , Fluorescent Dyes/chemistry , Adamantane/chemistry , Adamantane/metabolism , Binding, Competitive , Camphor/metabolism , Camphor 5-Monooxygenase/metabolism , Dansyl Compounds/chemistry , Dansyl Compounds/metabolism , Fluorescent Dyes/metabolism , Kinetics , Models, Molecular , Spectrometry, Fluorescence
7.
J Comput Aided Mol Des ; 15(7): 649-57, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11688945

ABSTRACT

Structure-based virtual screening techniques require reliable scoring functions to discriminate potential substrates effectively. In this study we compared the performance of GOLD, PMF, DOCK and FlexX scoring functions in FlexX flexible docking to cytochrome P450cam binding site. Crystal structures of protein-substrate complexes were most effectively reproduced by the FlexX/PMF method. On the other hand, the FlexX/GOLD approach provided the best correlation between experimental binding constants and predicted scores. Binding modes selected by the FlexX/PMF approach were rescored by GOLD to obtain a reliable measure of binding energetics. The effectiveness of the FlexX/PMF/GOLD method was demonstrated by the correct classification of 32 out of the 33 experimentally studied compounds and also in a virtual HTS test on a library of 10,000 compounds. Although almost all the available functions were developed to be general, our study on cytochrome P450cam substrates suggests that careful selection or even tailoring the scoring function might increase the prediction power of virtual screens significantly. The FlexX/PMF/GOLD methodology was tested on cytochrome P450 3A4 substrates and inhibitors. This preliminary study revealed that the combined function was able to recognise 334 out of the 345 compounds bound to 3A4.


Subject(s)
Camphor 5-Monooxygenase/metabolism , Drug Design , Binding Sites , Camphor 5-Monooxygenase/antagonists & inhibitors , Computer Simulation , Crystallography, X-Ray , Drug Evaluation, Preclinical , Energy Metabolism , Humans , In Vitro Techniques , Software , Substrate Specificity
8.
Xenobiotica ; 26(7): 723-53, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8819302

ABSTRACT

1. Using a novel amino acid sequence alignment, proteins of the CYP1A subfamily have been produced from the CYP102 crystal structure template via residue replacement and energy minimization procedures. 2. Known substrates and inhibitors of CYP1A1 and CYP1A2 are shown to fit their respective active sites via key interactions with complementary amino acid residues. Substrates used in the modelling studies include: caffeine, PhIP, oestradiol, 2,4- and 2,5-diaminotoluenes, Glu-P-1, phenacetin, acetanilide, 7-methoxy and 7-ethoxyresorufins, 11-methyl cyclopenta[a]phenanthren-17-one, 7-ethoxycoumarin, aflatoxin B1, benzo[a]pyrene, benzo[a]pyrene-7,8-diol and 1'-hydroxy 3-methylcholanthrene. 3. A number of aspects relating to CYP1A substrate specificity and metabolism can be explained in terms of the enzyme models, as it is found that key interactions with active site amino acid residues direct CYP1A-mediated metabolism in the known positions.


Subject(s)
Camphor 5-Monooxygenase/chemistry , Cytochrome P-450 CYP1A1/chemistry , Cytochrome P-450 CYP1A2/chemistry , Models, Molecular , Sequence Alignment , Amino Acid Sequence , Animals , Binding Sites , Camphor 5-Monooxygenase/antagonists & inhibitors , Camphor 5-Monooxygenase/metabolism , Cytochrome P-450 CYP1A1/antagonists & inhibitors , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP1A2 Inhibitors , Enzyme Inhibitors/metabolism , Humans , Mice , Molecular Sequence Data , Molecular Structure , Rabbits , Rats , Substrate Specificity , Trout
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