Pteroylpoly(gamma-glutamate) synthesis by Corynebacterium species. Purification and properties of folypoly(gamma-glutamate) synthetase.
Folylpolyglutamate synthetase was purified 7000-fold from extracts of Corynebacterium sp. The final preparation, which was greater than 95% pure, had a monomer molecular weight of 53,000. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to a variety of reduced pteroate and reduced pteroylmono-, di-, and triglutamate substrates with the concomitant production of ADP and Pi. Although the specificity for the folate substrate was wide, the pteroate and pteroylmonoglutamate substrates were utilized much more effectively than the polyglutamate derivatives. The most effective substrates were tetrahydropteroate, tetrahydrofolate, and 5,10-methylene-tretrahydrofolate. The most effective diglutamate substrate was 5,10-methylene-tetrahydropteroyldiglutamate. Addition of more than one glutamate moiety was only observed with tetrahydropteroate and 5,10-methylene-tetrahydropteroylmono- and diglutamates as substrates. The enzyme exhibited a preference for ATP as the nucleotide substrate. dATP was almost as effective while UTP and CTP were less effective substitutes. The specificity for L-glutamate appeared to be absolute. Enzyme activity was maximal at about pH 10 and exhibited an absolute requirement for a monovalent cation, of which K+ was the most effective. Preliminary studies suggest that the active form of the enzyme may be a dimer and that K+ may be required to effect dimerization.
D000255:Adenosine Triphosphate; D002268:Carboxypeptidases / Q000378:metabolism*; D002414:Cations, Monovalent; D003352:Corynebacterium / Q000201:enzymology*; D005492:Folic Acid / Q000031:analogs & derivatives*; D007700:Kinetics; D011624:Pteroylpolyglutamic Acids / Q000096:biosynthesis*; D013379:Substrate Specificity; D011623:gamma-Glutamyl Hydrolase / Q000302:isolation & purification / Q000378:metabolism*
D002414:Cations, Monovalent; D011624:Pteroylpolyglutamic Acids; D000255:Adenosine Triphosphate; D005492:Folic Acid; D002268:Carboxypeptidases; D011623:gamma-Glutamyl Hydrolase