Synthesis of (6R,11S)- and (6R,11R)-5,10-Methylene[11-¹H²H]tetrahydrofolate. Stereochemical Paths of Serine Hydroxymethyltransferase, 5,10-Methylenetetrahydrofolate Dehydrogenase, and Thymidylate Synthetase Catalysis

The stereochemical courses of the reactions catalyzed by serine hydroxymethyltransferase (EC 2.1.2.1) and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) have been determined. The method employed was to prepare the labeled intermediates (6R,11R)- and (6R,11R)-5,10-methylene[l l-¹H,²H]tetrahydrofolate by stereospecific chemical reduction and to determine their absolute stereochemistry at Cl 1 by the measurement of proton nuclear Overhauser enhancements (NOE's). Correlation of these results with those obtained by generating (6R)-5,10-methylene[l l^-1H,²H]tetrahydrofolate enzymatically from (3S)- and (3R)-[3^-1H,²H]serine with the transferase and from (6R)-5,10-methenyl[ll-²H]tetrahydrofolate via the dehydrogenase delineated the stereochemical paths of both enzymes. Serine hydroxymethyltransferase converts (3S)- and (35)-[3^-1H,²H]serine to (65,1 15)- and (6R,11R)-5,10-methylene[l l^-1H,²H]tetrahydrofolate, respectively, and the dehydrogenase stereospecifically removes H_R. These results also were correlated with those obtained by Tatum et al. [Tatum, C. M.; Vederas, J.; Schleicher, E.; Benkovic, S. J.; Floss, H. G. J. Chem. Soc. Chem. Commun. 1977, 218–220] on thymidylate synthetase (EC 2.1.1.b) to further delineate the stereochemical course of the one carbon unit transfer and accompanying reduction catalyzed by that enzyme.