Introduction
Trimethylglycine, also known as betaine, is found in microorganisms, animals, and plants. In most organisms it is biosynthesized by the oxidation of choline. Interest betaine metabolism has increased because elevated levels of plasma total homocysteine (tHcy) in blood is associated with certain vascular diseases. Therapies focused on reducing tHcy due to cystathionine β-synthase (CBS) deficiency include high levels of its cofactor pyridoxal phosphate. But approximately 40% of patients with CBS are unresponsive to pyridoxine therapy. For this reason, folic acid and betaine have been used to stimulate the remethylation of homocysteine (Hcy) to methionine (Met) by 5-methyltetrahydrofolate methyltransferase and betaine-homocysteine methyltransferase (BHMT), respectively. Betaine stimulation of BHMT is only partially effective for several reasons such as: DMG accumulation, which further inhibits the BHMT reaction, or Met accumulation, leading to hypermethioninemia.
Searching for other effective alternative treatments that can lower tHcy to a normal range is key. In vitro studies have shown that sulfonium analogs of betaine can be used as methyl donor substrates for the BHMT reaction, but it is not known if they would be effective treatments for human homocystinuria. Previous studies showed that sulfonium analogs of betaine, such as dimethylsulfonioacetate (DMSA)35 and
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dimethylsulfoniopropionate (DMSP), could be used by the BHMT reaction. Now, we want to evaluate the possibility that they could serve as treatment with homocystinuric patients if they can be shown to better than betaine as methyl donor substrates for the BHMT enzyme. Our objective is to determine the Michaelis constants (Km) for betaine
and the different sulfonium methyl donors for the BHMT reaction. First, we want to determine the Km of betaine because there is a discrepancy around its Km in the
literature. A broad range of values have been reported despite the fact that the mammalian BHMT enzymes share greater than 95% amino acid identity. It is possible that this discrepancy is due to experimental error, most likely to an inappropriate over consumption of betaine when assayed at low substrate concentrations (non-initial rate conditions). As depicted in figure 3, the lines represent experimentally determined initial rate velocity rates versus substrate concentration. Because enzyme-catalyzed reactions are saturables, their rates of reaction do not give a linear response to increasing of substrate. The most common error in the determination of Km values is an over consumption of substrate when tested at very low levels resulting in non-initial rate conditions. Our hypothesis is that some values that have been reported in the literature are low because of this unintentional error (Fig 3. 1; blue curve represents the over consumption of substrate, and the green curve a normal curve at initial rate).
Also, it would be to know how well the enzyme will use the other substrates, DMSA and DMSP. The inhibitory properties of the demethylated products of these sulfonium compounds, MTA and MTP, respectively, are now known. So far, MTP had the highest IC50 value, indicating it is not a potent inhibitor. However having a higher
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as well as betaine. We want to define the maximal catalytic activity (turn over, Kcat) of
these methyl donors so we can determine their catalytic efficiencies (Kcat/Km). Due to
time limitations and technical difficulties, this research only reported data for the initial velocity rate for betaine using purified human BHMT, rat and pig liver BHMT.
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Materials and Methods
Reagents
D, L-homocysteine thiolactone, Dowex 1x4, dimethyl-glycine (DMG) and β- mercaptoethanol were obtained from Sigma-Aldrich (St. Louis, MO). Bovine serum albumin (BSA) were obtained from Thermo Fisher Scientific (Pittsburgh, PA). Rat liver was obtained from Innovative Research Inc. (Novi, MI). Pig liver was a gift from Dr. L. Rund, Department of Animal Sciences, University of Illinois.
Initial Rate Kinetics of methyl donors
The BHMT standard assay was performed as described in chapter 2 with some modifications. Kinetic assays for all three enzymes (purified human, pig and rat liver) were conducted using the varying levels of betaine. Each assay contained [14C-methyl]-
betaine (0.5 µCi) and total betaine was varied from 0.25 to 20 mM. All the experimental data were obtained at fixed Hcy concentration (saturate levels, 100mM) and varying concentrations of betaine. To determine the kinetic constants, the production of nmol of Met/h or the reaction velocity was calculated. All experiments were repeated at least three times and in duplicate for each betaine concentration. The Km’s were estimated by
plotting initial rate data according to the method of Hanes (1932) using a simple linear regression (r2 > 0.97). To compare the results GraphPad Prism 6.04 was also used. The ratio of the initial substrate concentration [S] to the reaction velocity is plotted against [S].
45 Data Analysis
The data obtained were analyzed using one way analysis of variance to compare experimental to control values. Date are expressed as the mean ± standard deviation. Comparisons between groups were performed using least significant difference (LSD) test. Differences were considered significant at p<0.05. Results with different letter indicates significant difference (Fig 3.2).
46 Results
The results of these experiments are showed in figures 3.2 and 3.3. There was no significant difference between the two methods but there was significant difference between the enzymes. Michaelis constants for each BHMT enzyme was 1.83±0.5 mM, 0.71±0.06 mM, and 0.54±0.06 mM for human, pig and rat, respectively. The Km for the
rat liver and pig liver BHMT enzyme showed no significant difference between each other but both were significantly different from the human enzyme. These values obtained are in the range that was expected compared to the reported values in the literature.
47 0 5 1 0 1 5 0 .0 0 .5 1 .0 1 .5 H u m a n B H M T - S a t u r a t io n C u r v e Km = 1 .6 m M [ b e t a in e ] n m o l o f m e th io n in e /h o u r 0 5 1 0 1 5 0 .0 0 .5 1 .0 1 .5 2 .0 P ig B H M T - S a t u r a t io n C u r v e Km = 0 .7 2 m M [ b e t a in e ] n m o l o f m e th io n in e /h o u r 0 5 1 0 1 5 0 .0 0 .5 1 .0 1 .5 2 .0 R a t B H M T - S a t u r a t io n C u r v e Km = 0 .5 5 m M [ b e t a in e ] n m o l o f m e th io n in e /h o u r
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Figure 3.3 Kmbetaine Hanes Plot vs Graph Pad Prism
0 0.5 1 1.5 2 2.5 3
Human Pig Rat
Km (m M ) BHMT enzyme source Hanes Prism a a b b b b
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Discussion
It is important when doing enzyme kinetic measurements to do so at initial rate conditions. This means, ideally, that the consumption of substrate must be kept to a minimum so there is no accumulation of products and the substrate concentrations do not significantly change over the assay period. In this assay, the substrate (betaine) concentration was as low as 250 µM and at this low substrate concentration about 10% of the substrate was converted to product, and much less at the higher substrate concentrations tested. As betaine concentrations increased, Met production increased until saturation was reached. The data reported here indicate that the variation in the Km for betaine toward various BHMT enzymes is not nearly as great as reported in the literature.
The inability of betaine treatment to normalize tHcy in homocystinurics has led to the search for alternative methyl donors that might be better substrates for BHMT, as determined by high kcat values, and whose demethylated products might be weaker inhibitors of the enzyme. A primary objective of this study was to characterize two sulfonium analogs of betaine, DMSA and DMSP, to see if they have higher turnover rates compared to betaine, but this objective was not met due to technical difficulties and time allotted to this project. These studies will be continued by other personnel.
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Summary
This work demonstrated that the Km of betaine for the human, rat and pig BHMT
enzymes are similar and range between 0.5 to 1.8 mM. The differences in Km values
between these enzymes are relatively small compared to the values previously reported in the literature, and support the hypothesis that some experimental values were in error most likely due to the overconsumption of betaine at low substrate concentrations. A primary objective of this research was to characterize the kinetic properties of DMSA and DMSP as a first step to determine if they might be useful alternatives to betaine treatment for the treatment of homocystinuria, but due to time limitation and technical difficulties, it was only possible to determine the Km for betaine. Future studies will
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