Wayne L. Backes
Ph.D., West Virginia University, 1979
Cytochrome P450 refers to a superfamily of heme-containing enzymes involved in the metabolism of a wide variety of drugs, hydrocarbons, and other foreign compounds. This enzyme system is capable of metabolizing substrates of widely varying structure, with the basic process to insert an oxygen atom into the substrate molecule <Fig. 1>. The resulting product is more water soluble and generally more rapidly excreted; however, some of the resulting metabolites are reactive and can covalently bind to DNA, RNA and proteins. The bioactivation of foreign compounds by cytochrome P450 and some other xenobiotic-metabolizing enzymes can lead to toxicity, mutagenesis, carcinogenesis, and birth defects.
Figure 1 – Schematic of the cytochrome P450 enzyme system, showing the substrates and necessary co-factors in addition to the NADPH-cytochrome P450 reductase – P450 interaction that represents the simplest catalytically competent NADPH-mediated system
There are two reasons the cytochrome P450 system can metabolize a wide variety of substrates. First, the active site of cytochrome P450 is relatively non-selective and is capable of accommodating substrates of diverse chemical structures. The second reason for its broad selectivity is that there are many different P450 enzymes, each with their own characteristic substrate selectivities. Each of the P450 enzymes is independently regulated at least in part, by nutritional status, and prior exposure to foreign compounds.
The enzymes of the cytochrome P450 system do not act alone, but are part of an electron transport chain found in the endoplasmic reticulum. P450 requires an interaction with NADPH-cytochrome P450 reductase, a flavoprotein that is necessary for the transfer the reducing equivalents to P450. Other proteins that interact with those of the P450 system include cytochrome b5, NADH-cytochrome b5 reductase, and heme oxygenase.
Figure 2 – Schematic of the competition among P450 enzymes for limiting reductase found in the microsomal membrane. In liver microsomes the level of P450 exceeds that of the reductase by a factor of 10:1 to 20:1. When considering the fact that there are many different P450 enzymes present, there is the possibility for many different possible types of interactions.
An important characteristic of the P450 system that can influence its catalytic effectiveness is that, in most tissues, P450 is present in a large molar excess over the reductase. This creates conditions where a particular P450 enzyme will need to effectively compete with other P450 enzymes for the available reductase or be metabolically silent. The resulting metabolism of a foreign compound by the P450 system will depend on which P450 enzymes most effectively receive electrons from the reductase. A diagram of the problem is shown <Fig. 2>, where multiple P450 enzymes and other enzymes must compete for the reductase.
Figure 3 – Different mechanisms for the interaction of two P450 enzymes with reductase. When reductase concentrations are limiting, the ability of an individual P450 to compete for reductase will influence its catalytic effectiveness. Panel A shows a random distribution pattern for the reductase between two P450 isozymes. Under these conditions, both isozymes would be equally effective at competing for the reductase. Panel B shows the segregation of reductase to one of the P450 isozymes. Under these conditions, one enzyme (in this case 1A2) more effectively competes for the reductase. Panel C shows the formation of 1A2-2B4 complex that has altered binding characteristics when compared to the simple binary complexes. According to this model, the 1A2-2B4 complex preferentially binds reductase on the 1A2 moiety. This interaction differs from the type expected in the simple binary system (containing only reductase and a single P450), and can significantly influence the activity of this enzyme.
We are currently examining the organization of these proteins in order to explain how they might interact in the endoplasmic reticulum <related references>. Using purified CYP1A2 and CYP2B4 and reconstituting them with NADPH-cytochrome P450 reductase, we have shown that the presence of one P450 enzyme can influence the behavior of another P450. There are several possible mechanisms for the interaction of different P450 enzymes and reductase in the membrane <Fig. 3>. Our current data indicate different modes of interaction when different substrates are present. In studies where 7-pentoxyresorufin is the substrate, the data are consistent with the formation of a 1A2-2B4 complex where the catalytic characteristics of the reductase-1A2-2B4 complex differ from those of the simple binary complexes. More specifically, the addition of CYP1A2 to a reconstituted system containing CYP2B4 and subsaturating reductase leads to a dramatic inhibition in 7-pentoxyresorufin dealkylation (PROD). This inhibition is greater than that expected from a simple competition between CYP1A2 and CYP2B4 for the reductase, and is consistent with a model where the P450 enzymes form a complex that has an altered ability to associate with the reductase <see Fig. 3C>.
Figure 4 – Effect of ionic strength on the interaction among reductase, CYP2B4 and CYP1A2. At low ionic strength, the proteins can associate by electrostatic interactions, leading to the types of complexes shown; however, as the ionic strength is increased (click on image to begin animation), these electrostatic interactions can be disrupted. Complexes between CYP1A2 and CYP2B4 are diminished, leading to a decrease in the affinity of the reductase for CYP1A2. The result is the disruption of the CYP1A2-CYP2B4 complexes, which causes the dissociation of some reductase-CYP1A2 complexes in favor of the formation of reductase-CYP2B4 (click image to repeat animation).
We tested the CYP1A2/CYP2B4/reductase system to determine if these proteins interacted by charge-pairing. Many interactions among proteins are electrostatic in nature, with positively charged amino acids from one protein interacting with negatively charged amino acids on the other. Such electrostatic interactions can be disrupted by increasing the ionic strength of the buffer system. Our current data supports the concept of electrostatic interactions in that the large inhibition of PROD was reversed by at elevated ionic strength <Fig. 4>. This effect has also been shown for CYP1A2/CYP2E1/reductase reconstituted systems.
- Medical Pharmacology
- Interdisciplinary Graduate Course – Renal and Gastrointestinal Systems
- Responsible Conduct of Research
- Backes, W.L. and Reker-Backes, C.E. (1988) The Effect of NADPH Concentration on the Reduction of Cytochrome P-450 LM2. J. Biol. Chem. 263, 247-253.
- Backes, W.L., and Eyer, C.S. (1989) Cytochrome P-450 LM2 Reduction: Substrate Effects on the Rate of Reductase-LM2 Association. J. Biol. Chem. 264, 6252-6259.
- Causey, K.M., Eyer, C.S. and Backes, W.L. (1990) Dual Role of Phospholipid in the Reconstitution of Cytochrome P-450 LM2-Dependent Activities. Mol. Pharmacol. 38, 134-142.
- Eyer, C.S. and Backes, W.L. (1991) Relationship Between the Rate of Reductase - Cytochrome P450 Complex Formation and the Rate of First Electron Transfer. Arch. Biochem. Biophys. 293, 231-240.
- Cawley, G.F., Batie, C.J., and Backes, W.L. (1995) Substrate-Dependent Competition of Different P450 Isozymes for Limiting NADPH-Cytochrome P450 Reductase. Biochemistry 34, 1244-1247.
- Backes, W.L., Batie, C.J., and Cawley, G.F. (1998) Interactions Among P450 Isozymes: Evidence for the Existence of a 2B4-1A2-Reductase Complex with Altered Catalytic Function. Biochemistry 37, 12852-12859.
- Backes, W.L., Zhang, S., and Cawley, G.F., (2001) Evidence Supporting the Interaction of CYP2B4 and CYP1A2 in Microsomal Preparations. Drug Metabol. Dispos. 29, 1529-111534.
- Backes, W.L., and Kelley, R.W., (2003) Organization of Multiple Cytochrome P450s and NADPH-cytochrome P450 Reductase in Lipid Membranes, Pharmacol. Ther., in press.
- Cheng, Dongmei, Kelley, R.W., Cawley, G.F., and Backes, W.L. (2004) High Level Expression of Recombinant Rabbit Cytochrome P450 2E1 in Escherichia coli C41 and Its Purification. Prot. Express. Purific. 33, 66-71.
- Kelley, R.W., Reed, J.R. and Backes, W.L. (2005) Effects of Ionic Strength on the Functional Interactions between CYP2B4 and CYP1A2. Biochemistry, 44, 2632-2641.
- Backes, W.L., Kelley, R.W., Cheng, D., and Reed, J.R. (2005) How are NADPH-cytochrome P450 Reductase and Multiple Cytochromes P450 Organized in Membranes? Proceedings from the 14th International Conference on Cytochrome P450: Biochemistry, Biophysics and Bioinformatics 163-170.
- Reed, J.R., Kelley, R.W., and Backes, W.L. (2006) An Evaluation of Methods for the Reconstitution of Cytochromes P450 and NADPH P450 Reductase into Lipid Vesicles. Drug Metab. Dispos. 34, 660-666.
- Cormier, S.A., Lomnicki, S., Backes, W., and Dellinger, B. (2006) Origin and Health Impacts of Emissions of Toxic By-Products and Fine Particles from Combustion and Thermal Treatment of Hazardous Wastes and Materials. Environmental Health Persp. 114, 1-10.
- Kelley, R.W., Cheng, D., and Backes, W.L. Heteromeric Complex Formation Between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions. Biochemistry (in press).