Purification and properties of Lactobacillus casei folylpoly-gamma-glutamate synthetase.

摘要: Folylpolyglutamate synthetase was purified 200,000-fold from extracts of Lactobacillus casei. The homogeneous protein a monomer Mr = 43,000. enzyme catalyzed MgATP-dependent addition glutamate to 5,10-methylene-tetrahydropteroylmono-, di-, and triglutamate substrates metabolized (6R)-5,10-methylene-tetrahydro[3H]folate the tetraglutamate derivative. Other folate derivatives were poor or lacked activity. specificity nucleotide site wide. magnesium salts dATP, GTP, ITP, UTP effective alternate for reaction. binding very narrow. Of wide variety analogs tested, only L-homocysteate 4-fluoroglutamate demonstrated affinity enzyme. Kinetic studies consistent with an ordered Ter mechanism MgATP first enzyme, second, last. order product dissociation ADP, product, Pi. This precludes sequential moieties enzyme-bound folate. Michaelis constants (6R)-5,10-methylene-tetrahydropteroyldiglutamate, most substrate, MgATP, L-glutamate 2.3 microM, 5.6 mM, 423 respectively. Adenosine 5'-(3-thio)triphosphate beta, gamma-methylene-ATP inhibitors reaction had higher affinities than ATP.