Title

Purification And Characterization Of Betaine: Homocysteine Methyltransferase From Human Liver

Date of Award

1980

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

Abstract

L-Methionine, when activated by ATP to form S-adenosyl-L-methionine, serves as the methyl donor for many biological methylations. In mammals, the methylation of homocysteine to reform methionine is accomplished by two alternate pathways. N('5)-Methyltetrahydrofolate is one methyl donor and betaine, derived from choline, is the other. N('5)-Methyltetrahydrofolate: L-homocysteine methyltransferase, called "methionine synthetase" has been highly purified and has been extensively studied. Betaine:L-homocysteine methyltransferase (BHMase) has been less thoroughly studied. Although the nomenclasture suggests that two enzymes, betaine:homocysteine methyltransferase (EC 2.1.1.5) and dimethylthetin:homocysteine methyltransferase (EC 2.1.1.3), catalyze the betaine-dependent reaction, a critical review of the literature suggests that there may not be two distinct enzymes. In order better to understand methionine metabolism in man, the purification of BHMase from human liver was undertaken.Two enzymes catalyzing the BHMase reaction have been isolated from human liver. The component with the major activity (designated BHMase I) has been purified to apparent homogeneity. The protein has an M(,r) of approximately 270,000 by gel filtration; gel electrophoresis in the presence of sodium dodecyl sulfate suggests that the enzyme is composed of six subunits of about 45,000 daltons each. No evidence was found for any chromophoric cofactors and the activity was not affected by the chelating agent ethylenediaminetetraacetic acid (EDTA). There is evidence that the purified oligomeric protein aggregates into higher order polymers; incubation with sulfhydryl reducing agents regenerated and hexameric oligomer.The second component, BHMase II, had about 20% of the total activity and was separated from BHMase I at an early step in the purification by adsorption to diethylaminoethyl-cellulose. BHMase II was similar to BHMase I in amino acid composition and M(,r); however sodium dodecyl sulfate gel electrophoresis demonstrated major bands corresponding to 32,000 daltons and none corresponding to 45,000 daltons. The possibility that BHMase II is an artifact resulting from proteolytic cleavage of BHMase I must be considered, especially since the human liver source of enzyme was obtained frozen and therefore would be expected to release proteolytic enzymes upon thawing.Inhibition studies using substrate analogues can often provide evidence as to the nature of the active site. A number of betaine/dimethylglycine analogues were discovered to inhibit BHMase I, which suggests that the specificity of binding for the hypothesized betaine-dimethylglycine site is rather broad. A positive charge was not absolutely required for inhibition, perhaps indicating that the reaction product dimethylglycine binds in the unprotonated state. Analogues of methionine or homocysteine did not inhibit strongly.

Keywords

Chemistry, Biochemistry

Link to Full Text

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