Binding of the Folate Substrate to 5-Methyltetrahydropteroyltriglutamate-Homocysteine Transmethylase
Year: 1970
Type: article
Abstract: Binding studies of the 14C-folate substrate to the purified 5-methyltetrahydropteroyltriglutamate-homocysteine transmethylase have been undertaken to elucidate the enzymatic mechanism of action. Investigations of the binding of dl5-methyl-14C-tetrahydropteroyltriglutamate to the transmethylase reveal that one isomer of the intact folate substrate is bound to the enzyme in a reversible reaction at 0° or 25°. Eighty per cent of the radioactivity in the isolated enzyme-14C complex is converted to methionine on the addition of homocysteine. The maximum amount of binding observed is equivalent to 0.76 mole of 5-methyl-14C-tetrahydropteroyltriglutamate per mole of enzyme. The dissociation equilibrium constant of the enzyme-substrate complex has been calculated to be 4.5 x 10-7m in Tris buffer, and there is 0.96 substrate-binding site per molecule of enzyme. Evidence is presented that the binding of the folate substrate to the transmethylase occurs at the catalytic site of the enzyme or at a site with similar characteristics. The substrate specificity of the binding reaction is also consistent with that of the catalytic reaction. 5-Methyl-14C-tetrahydropteroyl-α-glutamylglutamate binds to the transmethylase 13 times less effectively than the triglutamate substrate, and 5-methyl-14C-tetrahydropteroylglutamate binds less effectively than the α-diglutamate derivative of the substrate. Furthermore, it has been concluded that the simultaneous removal of the methyl group on nitrogen atom 5 and oxidation of the 5, 6, 7, and 8 positions of the 5-methyltetrahydropteridine ring do not affect the binding of a folate compound to the enzyme. It has also been determined that the pteroate acid and glutamate moieties are essential for the binding of a folate compound to the transmethylase.
Source: Journal of Biological Chemistry
Authors: Carolyn D. Whitfield, Herbert Weissbach
Institution: National Institutes of Health
Language: English
Subfield: Rheumatology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1499412575