Primary structure and tetrahydropteroylglutamate binding site of rabbit liver cytosolic 5,10-methenyltetrahydrofolate synthetase

The primary sequence of 5,lO-methenyltetrahydrofolate synthetase from rabbit liver was determined by amino acid sequencing of the purified enzyme.The en- zyme contains 201 amino acid residues with a predicted mass of 22,779 Da.The enzyme is located in the cytosolic fraction of liver homogenates.Carbodiimide-activated 5-formyltetrahydropteroylmonoglutamate and the pentaglutamate form of the substrate both irreversibly inactivate the enzyme by forming a covalent bond to Lys-18.Non-activated 5-formyltetrahydropteroylpentaglutamate protected against this inactivation.Substrate specificity studies showed that increasing the number of glutamate residues from zero to five on 5-formyltetrahydropteroate results in a 2 order of magnitude increase in the affinity of the substrate for the enzyme but only a 3-fold increase in the value of V-. 5-Formyltetrahydropteroylglutamate(5-CHO-H4PteGlu,)' (clinically known as leucovorin) is used in cancer chemotherapy to either rescue patients from methotrexate toxicity or to enhance the effectiveness of 5-fluorouracil (1-5).5-CHO-H,PteGlu, exerts its effects by first being converted to 5,lO-CH+H,PteGlu, by an ATP-dependent reaction (Equation 1).The product 5,10-CH'-H4PteGlu, is converted to other reduced folate intermediates involved in l-carbon metabolism by the trifunctional enzyme C,-tetrahydrofolate synthase, serine hydroxymethyltransferase, and methylenetetrahydrofolate reductase (6-8).5-CHO-H4PteGlu, + MgATP + 5,10-CH+-H4PteGlu, + MgADP + P, (Eq. 1)Reaction 1 is catalyzed by 5,lO-methenyltetrahydrofolate synthetase (methenyl-THF synthetase), which has been puri-