Stereochemistry of Methylene Transfer Involving 5,10-Methylenetetrahydrofolate

The stereochemistry of the transfer catalyzed by rabbit liver serine transhydroxymethylase (EC 2.1.2.1) of the prochiral hydroxymethyl group from serine to tetrahydrofolate to form 5,10-methylenetetrahydrofolate was studied. Initial kinetic studies on labeled 5,10-methylenetetrahydrofolate showed that nonenzymatic racemization of the prochiral methylene center was buffer dependent and was slow under the conditions employed. Specifically tritiated (3R)- and (3S)-serines were employed to study the transfer reaction. Reactions were carried out under various conditions and the stereochemistry of the methylene carbon of the 5,10-methylenetetrahydrofolate produced was determined. The enzyme was shown to be partially stereospecific for this transfer reaction, proceeding with a loss of about 50% of the stereochemical purity of the transferred carbon center. Possible mechanistic interpretations of this finding are discussed.