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 degrees 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 microM) than for the monoglutamate (Km = 2 microM). The antifolate methotrexate has no inhibitory effect at concentrations up to 350 microM, while methotrexate pentaglutamate is a competitive inhibitor with a Ki = 15 microM. Similarly, dihydrofolate monoglutamate is a weak inhibitor with a Ki = 50 microM, while the pentaglutamate is a potent competitive inhibitor with a Ki of 3.8 microM. Thus, dihydrofolate and methotrexate pentaglutamates could regulate enzyme activity and help explain why leucovorin fails to rescue cells from high concentrations of methotrexate.
D000255:Adenosine Triphosphate / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D005492:Folic Acid / Q000031:analogs & derivatives*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology; D008970:Molecular Weight; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism*; D013379:Substrate Specificity
D011624:Pteroylpolyglutamic Acids; D000255:Adenosine Triphosphate; D005492:Folic Acid; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C021557:5,10-methenyltetrahydrofolate synthetase; D008727:Methotrexate