what the *** is that?

    Introduction
    Metabolism is the transformation of compounds produced within (endogenous) and outside (exogenous or xenobiotic) of an organism and the consequences that occur as these compounds are transported and eliminated by biological systems (ISSX 1). Both MAO and CYP3A4 are enzymes that are involved in complex systems of metabolism that can be studied by their effects on reaction rates of substrate to product conversion in order to find more information about how these systems work.
    Monoamine oxidase (MAO) is a type of flavoenzyme involved in redox reactions in biological systems (Castagnoli 2001; Kawai 1996). A flavin (see Figure 1) can be attached near the active site and aids the enzyme in catalytic activities (Kawai 1). The redox reactions catalyzed by flavoenzymes involve the conversion of an amine substrate to an aldehyde (Castagnoli 2001).

Amine-containing compounds, including many neurotoxins, neurotransmitters, and exogenous materials, such as pharmaceuticals, mutagens, and pollutants can undergo oxidative deamination reactions catalyzed by MAO (Castagnoli 2001, Lin 2001). MAO has been found to be located in the mitochondrial outer membrane in either A or B form; these forms are distinguished by differences in their selectivity (Castagnoli 2001).
Cytochrome P450s belong to a superfamily of heme containing proteins (Nelson 1). The cytochrome P450s can often be found in the endoplasmic reticulum (ER) and mitochondrial inner membrane (Lin 2001; Nelson 2003). These proteins are actually enzymes that catalyze the metabolism of endogenous substrates and xenobiotics (Lin 2001). Hydroxylation is the most common reaction catalyzed by cytochrome P450s, where a molecule of oxygen is used to oxidize the substrate and to form water (Nelson 2003). The process needs an electron donor, so NADPH is used in the ER while ferredoxin reductase and ferredoxin are used in the mitochondria. CYP3A4 is one cytochrome P450 that is involved in the metabolism of xenobiotics (Lin 2001).
The reactions with the enzyme can be thought to follow the following reaction scheme,

For the reaction scheme above, E is the participating enzyme, S is the substrate being used, ES the enzyme-substrate complex formed, and P is the final product (Clarke 1998). To describe the relationship between the substrate concentration and the metabolism rate, the Michaelis-Menten equation is often used for the P450 and MAO enzymes,

In the formula, [S] is the substrate concentrations. The rate of the reaction of metabolism is ν while Vmax is a constant that is the maximum value allowed for the rate. Km is another constant that describes the concentration of the substrate at which v is 50% of Vmax(Houston & Kenworthy 1999). When the metabolite kinetics follow the Michaelis-Menten model, then plotting ν vs. [S] gives a hyperbolic graph. In our experiments we will be assuming that the reactions will follow the Michaelis-Menten equation (Houston & Kenworthy 1999). CYP3A4 has been known to show non-hyperbolic metabolism curves and if the data show this behavior, we will adjust our model accordingly (Houston & Kenworthy 1999).
    For the proposed research, the specific aims are to establish methods for the assay of simultaneous MAO and CYP3A4 activity by studying the metabolic kinetics. There are five sets of reactions that are planned:
1.)    kynuramine and MAO A
2.)    7-benzyoxyquinolone and CYP3A4
3.)    kynuramine , MAO A and CYP3A4
4.)    7-benzyoxyquinolone, MAO A and CYP3A4
5.)    kynuramine, MAO A, 7-benzyoxyquinolone and CYP3A4
    These experiments will provide a platform for extensions to the studies of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) in hopes of contributing to research on Parkinson’s Disease that is being carried out at Virginia Tech University (Castagnoli 2001).

    Methods
    Human monoamine oxidase A (MAO-A) and human CYP3A4 (available from Gentest as SUPERSOMEStm produced by cDNA using a baculovirus expression system ) will be used in these experiments. Control SUPERSOMEStm will be used for a control comparison (also available from Gentest). Several typical MAO and CYP3A4 substrates will be studied, including kynuramine and 7-benzoxyquinolone, and these will be obtained from Sigma.
    The basic incubation method to be employed is as follows: The enzyme(s) will be mixed with the appropriate substrate(s) and incubated 37oC so that the reaction can occur (Gentest 1). Then to end at a particular time, an appropriate reagent is added to stop the reaction (Gentest 1). To obtain the metabolite solution free of protein, the sample will be centrifuged (Gentest 1). The solution after centrifuging will consist of an upper liquid layer (supernatant) above a solid layer, the solid layer consisting of mainly enzymes while the supernatant consisting of substrate, products, and solvent. Since we want to monitor the amount of product made over a certain time interval, the supernatant will be analyzed.
    A liquid chromatograph (Hewlett Packard 1090) will be used to monitor the progress of the various reactions. This apparatus works by having compounds present in a liquid mobile phase pass over a stationary phase (Braun 1987). The mixture is separated into its individual components based on the attraction of the components to a stationary phase (Braun 821). The distance a component travels in a certain amount of time can be used for qualitative analysis, while the amount of each component can be measured from the magnitude of the detector signal (Braun 1987). Thus we have a method to determine the rate of the reactions based on data on amount of each component of products based on time allowed for incubation.
    A fluorescence spectrometer (Varian Eclipse) will also be used to determine the amount of product made over a certain time interval. During fluorescence experiment, molecules absorb radiation and are excited from the ground state to a higher energy state. The molecules soon return to ground state but simultaneously emit light with an energy less than or equal to the energy absorbed. A portion of radiation that exits the cell containing the compound is measured by a detector (Braun 1987). After obtaining the data, a plot of the fluorescence intensity can be prepared and the concentration of product can be obtained (Braun 1987).

    Possible Results
     With kynuramine (K) and MAO A in one series of experiments and 7-benzyoxyquinolone (BQ) and CYP3A4 in another series of experiments, we expect to see rates that are characteristic of these particular enzyme-substrate systems. In each case, 4-hydroxyquinoline (HQ) is the expected product. With kynuramine dihydrobromide, MAO A and CYP3A4 in the same system, we expect MAO A to have the dominant metabolic rate since kynuramine is used as a standard substrate for this enzyme. With 7-benzyoxyquinolone, MAO A and CYP3A4 in the same system, we expect CYP3A4 to have the dominant metabolic rate since 7-benzyoxyquinolone is a characteristic substrate for this enzyme. For the paired enzymes with one substrate, if the enzymes operate independently then we anticipate the following relationship (Clarke 1998),

With all the reactants in the same system, we believe that we will see two rates that are characteristic of MAO A and CYP3A4. We will identify appropriate kinetic models to describe the observed kinetics for the following reaction pathways,

These are the currently hypothesized results for the five reaction systems to be studied . If differences should arise we will develop alternative kinetic models accordingly, in order that the method of studying various enzyme’s activities simultaneously can be improved.
The issue of how long the project will take is only left to question due to possible experimental complications. Working with enzymes and looking at their kinetics has become a new interest for Dr. Rutan’s laboratory at VCU and thus the basic methods for the assays must be identified and evaluated. There might be problems that occur in the procedure that could include stability of the supersomes, methods of solution preparation or collecting data that would have to be changed so that more appropriate data can be obtained. As mentioned previously, CYP3A4 kinetics might not follow the Michaelis-Menten equation. If this is the case then a sigmoidal or convex curve will be observed instead of the usual hyperbolic curve (Houston 1999). Such behavior is triggered by autoactivation or inhibition due to the substrate (Houston 1999). Thus a different means of expressing the kinetics will have to be derived and used. There also is a possibility that the experiment may go through with few or no complications and thus more substrates can be tested to if time permits. Testing some or all of the five reaction systems is the main goal for the summer, in hopes that during the school year the resulting techniques can be applied to tetrahydropyridines, which are of interest in the studies at Dr. Castagnoli’s laboratory (Castagnoli 2001).

1. Castagnoli, N., Dalvie, D., Kalgutkar, A., & Taylor, T. Interactions of Nitrogen     Containing Xenobiotics with Monoamine Oxidase (MAO) Isozymes A and B:       SAR Studies on MAO Substrates and Inhibitors. Chemical Research in     Toxicology, 14 (9), pp. 1139 -1162, 2001, http://pubs.acs.org/.
2. Clarke, S. In Vitro Assessment of Human Cytochrome P450. Xenobiotica, 28,(12), 1998, http://taylorandfrancis.metapress.com/.
3. Lin, Y., Lu, P., Mei, Q., Rodrigues, A., Rushmore, T., Sandig, G., Shou, M., & Tang, C. Substrate Inhibition Kinetics for Cytochrome P450-Catalyzed Reactions. Drug     Metabolism and Disposition, 29 (4), pp.368-374, April 2001, http://dmd.aspetjournals.org/.
4. Nelson,D. Cytochrome P450. Cytochrome P450 Homepage, April 23, 2003, http://drnelson.utmem.edu/CytochromeP450.html.
5. Parikh, S., Hanscom, S., Gagne, P., Crespi, C., & Patten, C. A Fluroescent-Based, High-Throughput Assay for Inhibitors of Human Monoamine Oxidase A and         B. Gentest. http://www.gentest.com/.
6. Houston, J. & Kenworthy, K. In Vitro-In Vivo Scaling of CYP Kinetic Data not Consistent with the Classical Michaelis-Menten Model. The American Society             for Pharmacology and Experimental Therapeutics. 28 (3), pp.246-254, November 1999, http://www.dmd.org.
7. Kawai, Y., Kunitomo, J., & Ohno, A. Atropisomeric Flavoenzyme Models with a Modified Pyrimidine. Kyoto: Institute for Chemical Research – ICR Annual             Report. 3, 1996, http://www.kuicr.kyoto-u.ac.jp/.
8. ISSX Constitution. International Society for the Study of Xenobiotics, http://www.issx.org/.
9. Human Monoamine Oxidase A (MAO-A). Gentest, http://www.gentest.com/.
10. Braun, R. Introduction to Instrumental Analysis. McGraw-hill, pp.316-346; 821-869,1987.

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