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Protein-protein and lipid-protein interactions in a reconstituted cytochrome P-450 dependent microsomal monooxygenase
1Institute for Protein Research, Osaka University, Japan.
Abstract:
NADPH-cytochrome P-450 reductase and cytochrome P-450, both purified from liver microsomes of phenobarbital-treated rabbits, were incorporated into dimyristoylphosphatidylcholine vesicles. The reduction of cytochrome P-450 by NADPH in the reconstituted vesicles proceeded in a biphasic fashion, and 70-80% of the absorbance change was associated with the fast phase. The Arrhenius plot of the apparent first-order rate constant of the fast-phase reduction showed a marked discontinuity around the phase transition temperature of the synthetic phospholipid; an almost 10-fold change in rate constant was associated with this discontinuity. It was, therefore, suggested that the reduction of cytochrome P-450 by reductase in this system was a diffusion-limited reaction controlled by the viscosity of the phospholipid membrane. The Arrhenius plot of overall drug monooxygenase activity catalyzed by the reconstituted vesicles showed a break but in a different way from that observed for the reduction of cytochrome P-450. This break was accompanied only by a change of the slope of the plot but not by a change in reaction rate. This difference in the two Arrhenius plots was attributed to that in the rate-limiting step of the two reactions. NADPH-cytochrome c reductase activity of the reconstituted vesicles, an activity catalyzed by the reductase alone, and cumene hydroperoxide dependent N-methylaniline demethylation activity catalyzed by cytochrome P-450 alone did not show any break in the Arrhenius plots.
Insights
The reduction of cytochrome P-450 by NADPH-cytochrome P-450 reductase in lipid vesicles is diffusion-limited, influenced by membrane viscosity. This contrasts with overall drug monooxygenase activity, suggesting different rate-limiting steps.
Area of Science:
- Biochemistry
- Membrane Biophysics
- Enzyme Kinetics
Background:
- NADPH-cytochrome P-450 reductase and cytochrome P-450 are key enzymes in drug metabolism.
- Reconstituting these enzymes into phospholipid vesicles allows for controlled study of their interactions.
- Understanding the physical and chemical factors influencing enzyme activity in membranes is crucial.
Purpose of the Study:
- To investigate the kinetics and rate-limiting steps of cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase within dimyristoylphosphatidylcholine vesicles.
- To explore the influence of phospholipid membrane properties, specifically phase transitions, on enzyme function.
- To compare the kinetic behavior of enzyme reduction with overall catalytic activity.
Main Methods:
- Purification of NADPH-cytochrome P-450 reductase and cytochrome P-450 from rabbit liver microsomes.
- Reconstitution of purified enzymes into dimyristoylphosphatidylcholine vesicles.
- Kinetic analysis using spectrophotometry and Arrhenius plots to determine reaction rates and activation energies at varying temperatures.
Main Results:
- Cytochrome P-450 reduction by NADPH in reconstituted vesicles exhibited biphasic kinetics, with 70-80% in a fast phase.
- The fast-phase reduction showed a marked discontinuity in its Arrhenius plot near the phospholipid phase transition, indicating diffusion limitation by membrane viscosity.
- Overall drug monooxygenase activity displayed a different break in its Arrhenius plot, suggesting a distinct rate-limiting step compared to enzyme reduction.
Conclusions:
- The reduction of cytochrome P-450 by NADPH-cytochrome P-450 reductase in this reconstituted system is primarily diffusion-limited, controlled by the viscosity of the phospholipid membrane.
- The rate-limiting step for overall drug monooxygenase activity differs from that of direct enzyme reduction, highlighting complex reaction mechanisms.
- Membrane fluidity significantly impacts the kinetics of membrane-bound enzyme systems.