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

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-H 4 PteGlu and 5-methyl-H 4 PteGlu 5 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-H 4 PteGlu 5 was a more effective substrate for the methyltransferase (K m ~ 4 μM) and reductase (K m ~ 3 μM), at low substrate concentrations, than (+)-5-methyl-H 4 PteGlu (K m ~ 13 μM and ~ 23 μM, respectively), although V values were lower. High levels of the pentaglutamate substrate inhibited the reductase reaction (K i ~ 40 μM). The unnatural (−)-5-methyl-H 4 PteGlu 1,5 diastereoisomers were not inhibitors of either enzyme.The results suggest that any 'methyl trap' operating at the monoglutamate level under conditions of vitamin B 12 deprivation would be at least as effective at the pentaglutamate level.