HUMAN-LIVER METHENYLTETRAHYDROFOLATE SYNTHETASE - IMPROVED PURIFICATION AND INCREASED AFFINITY FOR FOLATE POLYGLUTAMATE SUBSTRATES

Methenyltetrahydrofolate synthetase (5-formyltetrahydrofolate cyclodehydrase (cyclo-ligase) (ADP-forming) EC 6.3.3.2) catalyzes the ATP-and Mg2+-dependent transformation of 5-formyltetrahydrofolate (leucovorin) to 5,10-methenyltetrahydrofolate. The enzyme has been purified 49,000-fold from human liver by a two-column procedure with Blue Sepharose followed by folinate-Sepharose chromatography. It appears as a single band both on SDS-polyacrylamide gel electrophoresis (Mr 27,000) and on isoelectric focusing (pI = 7.0) and is monomeric, with a molecular weight of 27,000 on gel filtration. Initial-velocity studies suggest that the enzyme catalyzes a sequential mechanism and at 30.degree.C and pH 6.0 the turnover number is 1000 min-1. The enzyme has a higher affinity for its pentaglutamate substrate (Km = 0.6 .mu.M) than for the monoglutamate (Km = 2 .mu.M). The antifolate methotrexate has no inhibitory effect at concentrations up to 350 .mu.M, while methotrexate pentaglutamate is a competitive inhibitor with a Ki = 15 .mu.M. Similarly, dihydrofolate monoglutamate is a weak inhibitor with a Ki = 50 .mu.M, while the pentaglutamate is a potent competitive inhibitor with a Ki of 3.8 .mu.M. Thus, dihydrofolate and methotrexate pentaglutamates could regulate enzyme activity and help explain why leucovorin fails to rescue cells from high concentrations of methotrexate.