title: Purification and properties of Lactobacillus casei folylpoly-gamma-glutamate synthetase.
abstract: Folylpolyglutamate synthetase was purified 200,000-fold from extracts of Lactobacillus casei. The homogeneous protein was a monomer of Mr = 43,000. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to 5,10-methylene-tetrahydropteroylmono-, di-, and triglutamate substrates and metabolized (6R)-5,10-methylene-tetrahydro[3H]folate to the tetraglutamate derivative. Other folate derivatives were poor substrates or lacked activity. The specificity of the nucleotide site was wide. The magnesium salts of dATP, GTP, ITP, and UTP were effective alternate substrates for the reaction. The specificity of the glutamate binding site was very narrow. Of a wide variety of analogs tested, only L-homocysteate and 4-fluoroglutamate demonstrated affinity for the enzyme. Kinetic studies were consistent with an ordered Ter Ter mechanism with MgATP binding first to the enzyme, folate second, and glutamate last. The order of product dissociation from the enzyme was ADP, folate product, and Pi. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. The Michaelis constants for (6R)-5,10-methylene-tetrahydropteroyldiglutamate, the most effective folate substrate, MgATP, and L-glutamate were 2.3 microM, 5.6 mM, and 423 microM, respectively. Adenosine 5'-(3-thio)triphosphate and beta, gamma-methylene-ATP were inhibitors of the reaction and had higher affinities for the enzyme than ATP.
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010453:Peptide Synthases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005971:Glutamates; D046911:Macromolecular Substances; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase