Pentaglutamate Derivatives of Folate as Substrates for Rat Liver Tetrahydropteroylglutamate Methyltransferase and 5,10-Methylenetetrahydrofolate Reductase

Abstract

Tetrahydropteroylglutamate methyltransferase (EC 2.1.1.13) and 5,10-methylenetetrahydrofolate reductase (EC 1.1.1.68) were purified more than 100-fold from rat liver. The specificity of each enzyme for 5-methyl-H4PteGlu and 5-methyl-H4PteGlu5 did not change throughout the purification procedures.A comparison of enzyme properties as well as kinetic analysis showed that pteroylmono- and pentaglutamates were binding to the same enzyme in each case. There was no evidence of any pteroylpentaglutamate specific methyltransferase or reductase in rat liver.(+)-5-Methyl-H4PteGlu5 was a more effective substrate for the methyltransferase (Km ~ 4 μM) and reductase (Km ~ 3 μM), at low substrate concentrations, than (+)-5-methyl-H4PteGlu (Km ~ 13 μM and ~ 23 μM, respectively), although V values were lower. High levels of the pentaglutamate substrate inhibited the reductase reaction (Ki ~ 40 μM). The unnatural (−)-5-methyl-H4PteGlu1,5 diastereoisomers were not inhibitors of either enzyme.The results suggest that any 'methyl trap' operating at the monoglutamate level under conditions of vitamin B12 deprivation would be at least as effective at the pentaglutamate level.