Active site mechanics of liver microsomal cytochrome P-450

White, Ronald E.; McCarthy, Mary-Beth

doi:10.1016/0003-9861(86)90445-5

Cited by 39 publications

(23 citation statements)

References 26 publications

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“…By determining the K m of each P450 for toluene metabolism, we confirmed that both enzymes were close to saturation with substrate under the experimental conditions used. Interestingly, our measurement of the K m for toluene metabolism by CYP2B4 was about 15-fold smaller than the value previously reported for this substrate (41). This discrepancy is likely attributable to (1) differences in the methods for protein reconstitution, and (2) the addition of toluene as a methanolic solution in the previous study.…”

Section: Discussioncontrasting

confidence: 92%

“…Previous studies have shown that the catalytic characteristics of the P450s are extremely sensitive to reconstitution conditions, as the proteins need to be preincubated for at least 1 hr in order to stabilize the catalytic results (42). The duration of enzymes/lipid preincubation is unclear from the previous kinetic analysis of toluene metabolism (41). The second major variant is due to the use of organic solvents as vehicles for hydrophobic substrates.…”

Section: Discussionmentioning

confidence: 99%

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Interactions between Cytochromes P450 2B4 (CYP2B4) and 1A2 (CYP1A2) Lead to Alterations in Toluene Disposition and P450 Uncoupling

2013

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The goal of this study was to characterize the effects of CYP1A2•CYP2B4 complex formation on the rates and efficiency of toluene metabolism by comparing the results from simple reconstituted systems containing P450 reductase (CPR) and a single P450 to those using a mixed system containing CPR and both P450s. In the mixed system, the rates of formation of CYP2B4-specific benzyl alcohol and p-cresol were inhibited, whereas that of CYP1A2-specific o-cresol was increased, results consistent with the formation of a CYP1A2•CYP2B4 complex where the CYP1A2 moiety has higher affinity for CPR binding. Comparison of the rates of NADPH oxidation and production of hydrogen peroxide and excess water by the simple and mixed systems indicated that excess water formed at a much lower rate in the mixed system. The commensurate increase in the rate of CYP1A2-specific product formation suggested the P450•P450 interaction increased the putative rate-limiting step of CYP1A2 catalysis, abstraction of a hydrogen radical from the substrate. Cumene hydroperoxide-supported metabolism was measured to determine whether the effects of the P450•P450 interaction required the presence of CPR. Peroxidative metabolism was not affected by the interaction of the two P450s, even with CPR present. However, CPR did stimulate peroxidative metabolism by the simple system containing CYP1A2. These results suggest the major functional effects of the P450•P450 interaction are mediated by changes in the relative abilities of the P450s to receive electrons from CPR. Furthermore, CPR may play an effector role by causing a conformation change in CYP1A2 that makes its metabolism more efficient.

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Section: Discussioncontrasting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Interactions between Cytochromes P450 2B4 (CYP2B4) and 1A2 (CYP1A2) Lead to Alterations in Toluene Disposition and P450 Uncoupling

2013

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“…Data for P450-Mediated Hydroxylation of para-ToluenesNote: Molecular orbital calculations were carried out via the AM1 method[28,64,65].IP ϭ ionization potential (eV) for the hydrogen-abstracted species ∆H ‡ ϭ enthalpy change (kcal/mol) for formation of the hydrogen-abstrated species SA ϭ solvent accessible surface area (Å 2 ) of the Connolly surface ∆E ϭ E(LUMO) Ϫ E(HOMO), where E(LUMO) and E(HOMO) are the energies (eV) of the lowest unoccupied and highest occupied MOs, respectivelylog P ϭ logarithm of the n-octanol/water partition coefficient[83,84] k ϭ rate constant for P450-catalyzed hydroxylation in rabbit liver microsomes[85] K m ϭ apparent Michaelis constant for P450-catalyzed hydroxylation in rabbit liver microsomes[85] Source: Refs. 43, 60, and 86.Drug Metabolism Reviews Downloaded from informahealthcare.com by University of California San Francisco on 11/18/14For personal use only.…”

mentioning

confidence: 99%

Frontier Orbitals in Chemical and Biological Activity: Quantitative Relationships and Mechanistic Implications*

Lewis

1999

Drug Metabolism Reviews

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“…However, modelling of this procedure of necessity leads to a cor-rection term [,E -(eB+ c' K0 aL)/(l + KpaL)] which depends on the wavelength, resulting in a distortion of the difference spectrum. Similarly, no typical type-I peak was present in p-nitrotolueneinduced difference spectra of cytochrome P-450 LM2, corrected for the proper absorbance of the solute (White & McCarthy, 1986).…”

Section: Appendix Mathematical Analysis Of Difference Spectramentioning

confidence: 92%

Interaction of 7-n-alkoxycoumarins with cytochrome P-4502 and their partitioning into liposomal membranes. Assessment of methods for determination of membrane partition coefficients

Vermeir

Boens

Heirwegh

1992

Biochemical Journal

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A study was made of the binding of 7-ethoxy-, 7-n-propoxy-and 7-n-pentoxy-coumarin to cytochrome P-4502 reconstituted into large unilamellar liposomes composed of a mixture of egg L-a-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoyl phosphatidic acid (2:1:0.06, by weight). The apparent spectral dissociation constants QPP-increased linearly with increasing proteoliposomal concentration. When both cytochrome P-4502 and NADPH:cytochrome P-450 reductase were reconstituted into liposomes, the apparent Michaelis constants KmPP for 0-dealkylation of 7-methoxy-, 7-ethoxy-and 7-n-propoxy-coumarin showed a similar dependence on the proteoliposomal concentration. The results were in accordance with models for kinetic or equilibrium processes in biphasic systems containing membrane-bound catalytic or acceptor sites, in which a linear solute partition in the bilayer membrane is postulated. The methyl, ethyl and n-propyl ether were readily dealkylated. However, the O-dealkylation rate of 7-nbutoxycoumarin was low and became very small for longer alkyl ethers. Both the effective dissociation constants and effective Michaelis constants decreased with elongation of the alkyl side chain of the coumarins. From plots of the apparent dissociation constants and apparent Michaelis constants against the lipid volume fraction of the proteoliposomes, the membrane partition coefficients for several homologues were calculated. When protein-free liposomes were added to 7-n-alkoxycoumarin solutions, the fluorescence intensity of the coumarins decreased and eventually became negligible in the presence of an excess of liposomal material. On the assumption that the overall fluorescence can be ascribed exclusively to the fraction of 7-n-alkoxycoumarin molecules present in the aqueous phase, partition coefficients for liposomal accumulation of the test compounds could be determined directly. For several coumarin ethers, comparable values were derived for the membrane partition coefficients from binding, kinetic and fluorescence intensity measurements. The change in free energy per methylene group of the 7-n-alkoxycoumarins for partitioning between n-octanol and buffer was significantly different from the value for liposome partitioning.

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Active site mechanics of liver microsomal cytochrome P-450

Cited by 39 publications

References 26 publications

Interactions between Cytochromes P450 2B4 (CYP2B4) and 1A2 (CYP1A2) Lead to Alterations in Toluene Disposition and P450 Uncoupling

Interactions between Cytochromes P450 2B4 (CYP2B4) and 1A2 (CYP1A2) Lead to Alterations in Toluene Disposition and P450 Uncoupling

Frontier Orbitals in Chemical and Biological Activity: Quantitative Relationships and Mechanistic Implications*

Interaction of 7-n-alkoxycoumarins with cytochrome P-4502 and their partitioning into liposomal membranes. Assessment of methods for determination of membrane partition coefficients

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