|
Monoamine Oxidase (MAO) is present in all human cells and is responsible for the oxidative deamination of dietary amines and neurotransmitters. There are two
types of MAO – MAO A and MAO B – each with overlapping yet distinct physiological roles. The optimal temperature for MAO catalysis is unknown. In this study,
optimal MAO A and MAO B activity was determined by assaying the oxidation of para-trifluoromethyl-benzylamine (p-CF3-BA) at various temperatures. A
temperature dependence of p-CF3-BA oxidation by MAO A and MAO B was observed and the optimal temperature for activity determined to be 25°C and 35°
C for MAO A and B respectively.
The enzymes monoamine oxidase A and B are anchored into the outer membrane of the mitochondria. MAO catalyzes the oxidative deamination of
neurotransmitters and biogenic amines through utility of a bound FAD cofactor.1 Since MAO A has a role in metabolizing serotonin and MAO B functions to oxidize
dopamine, these enzymes are targeted for pharmacological benefits. Inhibiting these enzymes permit these neurotransmitters to exist in larger concentrations,
which leads to mood elevation thereby combating depression. Thus, extending insight into the properties of these enzymes can lead to the rational development
of inhibitor drugs. The general mechanism of MAO catalysis is shown in scheme 1.
The optimum temperature from which MAO A and MAO B are active is unknown. In this study, MAO A and MAO B was assayed by measuring the oxidation of
p-CF3-BA at various temperatures. The kinetic parameters determined at these different temperatures will reveal the optimal temperature of catalytic efficiency
for MAO.
Two chief kinetic parameters determined at each temperature are Vmax and KM. Vmax is the maximum velocity of a reaction in the presence of a given amount
of enzyme, whereas KM measures an enzyme’s affinity for a particular substrate. Calculating kcat/KM discloses the catalytic efficiency of an enzyme and a
substrate.
In this study, MAO A and B were assayed at 7 different concentrations for each temperature tested: 20, 30, 50, 100, 300, 500, and 800 µM. A cuvette containing
the p-CF3-BA reaction mixture was placed in a water bath to control temperature. The temperature of each cuvette was measured immediately prior to the initiation
of each assay. Absorbance changes over time were then measured spectrophotometrically at 234 nm, indicating oxidation of p-CF3-BA. The initial velocity of
these reactions was plotted as a function of substrate concentration, and the data fit to a hyperbola function using graphing software. This fit reveals the kinetic
parameters – Vmax and KM – for each temperature tested.
Figure 1. Rate dependence of p-CF3-benzylaldehyde oxidation by hMAO A upon [p-CF3-BA]. The graph illustrates dependence of p-CF3-benzylamine oxidation
upon substrate concentration. The data was fit to a hyperbola function.
Figure 2. Lineweaver-Burk plot of figure 1 data. The data in figure 1 was linearized to access if the data behaves according to classical Michaelis-menton kinetics.
Vmax and KM can be extrapolated from a graph of this manner and compared to the values obtained in figure 1.
Figure 3. Rate dependence of p-CF3-benzylaldehyde oxidation by hMAO B upon [p-CF3-BA]. The graph illustrates dependence of p-CF3-benzylamine oxidation
upon substrate concentration. The data was fit to a hyperbola function.
Figure 4. Lineweaver-Burk plot of figure 3 data. The data in figure 3 was linearized to access if the data behaves according to classical Michaelis-menton
kinetics.
Figure 5. Dependence of MAO A activity upon temperature. These data show the temperature optimum of amine oxidation for MAO A is approxamately 25ºC.
Figure 6. Dependence of MAO B activity upon temperature. These data show the temperature optimum of amine oxidation for MAO B is approxamately 35ºC.
In this study, our goal was to determine the optimal temperature for MAO A and MAO B activity. From Figures 1 and 3, the kinetic parameters Vmax and KM
were determined for each temperature by plotting the initial velocities of p-CF3-benzylamine oxidation by MAO. The kinetic parameters obtained from linearizing
the data (Figures 2 and 4) further bolsters the validity of these values. Using these data, the catalytic efficiency (kcat/KM) was determined and plotted as a function
of temperature. Thus, the optimal temperatures of activity for MAO A and MAO B were obtained.
I would like to thank Dr. Dale Edmondson for the opportunity to work in his laboratory. A very special thank you to Dr. Francisco Cruz , my co-mentor, and all of the
members of the Edmondson Lab group. And lastly, thank you to the Emory University SURE Program for this wonderful research opportunity.
This research was supported by NIH Grant # GM-29433 and the SURE Program National Science Foundation Award # 0450303, sub-award #1-66-606-63
to Emory University.
1 Edmondson, D. and A. Mattevi. Structure and Mechanism of Monoamine Oxidase. Current Medical Chemistry. 2004, 1983-1993.
2 Kleinsmith, L. and V. Kish. Principles of Cell and Molecular Biology. 2nd ed. 39-47.
Have you ever had too many tests to study for in one night? Has this feeling of stress sent you into depression because you felt like it simply could not be done?
Well, my research dealt with the enzymes that cause you to feel depressed. The enzymes are known as Monoamine Oxidase A and B. In my study, I was trying
to find at which temperature these enzymes worked the best, thus figuring out the best way to slow them down and reduce depression.
UV-spectroscopy
MAO A and B, temperature dependence, amine oxidation, p-Cf3-benzylamine oxidation, MAO A studies
|
