Kinetic characterization of methyl donor substrates and inhibitors of human, pig, and rat liver betaine homocysteine S-methyltransferase (BHMT)

Homocysteine;Methionine;Betaine;Enzymology;Kinetic

Aubourg, Nadine

关键词: Homocysteine;    Methionine;    Betaine;    Enzymology;    Kinetic;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/78799/AUBOURG-THESIS-2015.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Betaine-homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine to form dimethylglycine and methionine, respectively. BMHT is primarily expressed in the liver and kidney of mammals. BHMT catalyzes an ordered bi bi reaction where the first product released, dimethylglycine, can compete for the betaine binding site and inhibit homocysteine utilization. There is considerable interest in the regulation of homocysteine metabolism since even moderate elevations in plasma total homocysteine have been established as an independent risk factor for the development of vascular diseases and thrombosis. In people with homocystinuria, treatment using supplemental betaine showed a significant decrease in total plasma homocysteine but does not lower homocysteine levels within the normal range, and the moderate levels that remain are highly correlated with vascular disease. Therefore, the BHMT catalyzed reaction is a target for the treatment of homocystinuria. Finding alternative methyl donors for the BHMT reaction that following methyl transfer have less potent inhibitory properties than dimethylglycine are desired. Sulfonium analogs of betaine are considered for this research. The main objectives of this research were to determine the inhibitory properties of the demethylated product of betaine and its sulfonium analogs and also to determine the Michaelis constants in order to use them as alternative methyl donors for the BHMT reaction in homocystinuric patients. The methyl donor substrates used are betaine, dimethylsulfonioacetate, dimethylsulfoniopropionate, and their respective demethylated products are dimethylglycine, methylthioacetate and methylpropionate. Dimethylglycine had the lowest IC50 values, ranging from 28 to 35 µM for all three enzymes. Methylthioacetate had IC50 values ranging from 65 to 106 µM, and values for methylthiopropionate ranged from 400 to 800 µM. There was no significant difference between the IC50 values obtained for the different enzymes when assayed in the presence of DMG or MTA, but for MTP the IC50 values were significantly different from one enzyme to another.Kinetic studies for betaine was conducted for all three enzymes. The Km of betaine varied from 1.8±0.5 mM, 0.7±0.06, and 0.5±0.06 mM for overexpressed human BHMT, pig and rat liver BHMT, respectively. We are unable to determine the Km for DMSA and DMSP. Further studies as the catalytic efficient (Kcat) for all three substrates are needed in other to determine which one could be a better alternative treatment for homocystinuria.