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 enzymes are involved in complex
metabolic systems. Studying the reaction rates (time require to convert
substrate
to product) of these enzymes can provide 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 convert amine
substrates to aldehydes (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
is in the mitochondrial outer membrane in either A or B form; these
forms are distinguishable 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,
where a molecule of oxygen is used to oxidize
the substrate and to form water, is the
most common reaction catalyzed by cytochrome P450s (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 an example of a 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 shows 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 their metabolic kinetics. Five sets of reactions 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 spun in a centrifuge (Gentest 1). Which
will give a solution with an upper liquid layer (supernatant) above a
solid layer, the solid layer will mainly consist of enzymes while the
supernatant consisting of
substrate, products, and solvent. Since we want to monitor the amount
of product during a given 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 the amount of products.
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).
