Betaine:homocysteine methyltransferase from rat liver: purification and inhibition by a boronic acid substrate analog.
Betaine:homocysteine methyltransferase (BHMT) from rat liver has been highly purified by an efficient procedure requiring only two chromatographic steps: Sephadex G-100 chromatography and fast protein liquid chromatography chromatofocusing. A 170-fold purification and 7.5% overall yield were achieved. Chromatofocusing yielded three active forms of BHMT with pI values near 8.0, 7.6, and 7.0. The subunit molecular weight of each active form is 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native enzyme has a molecular weight of 270,000 as determined by exclusion chromatography. The stability of the purified enzyme was found to be potentiated by the presence of 1 mM dimethylglycine and 1 mM homocysteine. Boronate analogs of betaine (pinanyl N,N,N-trimethylaminomethaneboronate) (4) and dimethylglycine (pinanyl N,N-dimethylaminomethaneboronate) were synthesized from pinanyl iodomethaneboronate (3) and trimethylamine or dimethylamine, respectively. The free acid of the betaine analog (5) was reversibly generated from (4). The inhibition of BHMT by (5) appears competitive with a Ki = 45 microM. Since the Km for betaine measured with the purified enzyme is near 0.1 mM, the boronic acid analog of betaine appears to function effectively as a substrate analog inhibitor of BHMT. The analog does not appear to act as a methyl donor to homocysteine when (5) is substituted for betaine in the enzyme reaction. In addition, an enzyme assay based upon C3-cyano reverse phase HPLC detection of the o-phthalaldehyde derivative of methionine was developed as an alternative to the standard radiochemical assay. Betaine:homocysteine methyltransferase in the picomole range can be quantitated using this assay as indicated by a linear response of enzyme activity to protein concentration.
Department of Chemistry and Biochemistry, San Francisco State University, California 94132.
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Lee
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Cava
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Amiri
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Ottoboni
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Lindquist
D000818:Animals; D001622:Betaine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D050940:Betaine-Homocysteine S-Methyltransferase; D001897:Boronic Acids / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004795:Enzyme Stability; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012521:Sarcosine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology; D013379:Substrate Specificity
D001897:Boronic Acids; D001622:Betaine; C025138:dimethylglycine; D008780:Methyltransferases; D050940:Betaine-Homocysteine S-Methyltransferase; C494550:Bhmt protein, rat; D012521:Sarcosine