Enzymatic synthesis of folate and antifolate polyglutamates with Escherichia coli folylpolyglutamate synthetase.
Escherichia coli folylpolyglutamate synthetase was used to synthesize micromole quantities of polyglutamyl conjugates of folic acid, methotrexate, and other analogs of folic acid. The products of the enzymatic reactions were purified by semipreparative C18 HPLC. The position of each amide linkage (gamma or alpha carboxyl) in the polyglutamated products was determined by limited and exhaustive hydrolyses with hog kidney folylpolyglutamate hydrolase and with yeast carboxypeptidase Y. Under standard reaction conditions, the E. coli enzyme added up to five glutamyl residues to each monoglutamated substrate, primarily at the gamma carboxyl position. Thus, an enzyme which naturally adds only two glutamates to naturally occurring folates can be used synthetically to make higher polyglutamates of a wide range of synthetic substrates. The products of the reactions are valuable tools for the study of the metabolism of antifolate drugs as well as metabolic reactions involving folate cofactors.
Hanlon
M H
MH
Department of Molecular Genetics and Microbiology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Ferone
R
R
Weaver
K
K
Ray
P
P
D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005493:Folic Acid Antagonists / Q000138:chemical synthesis* / Q000302:isolation & purification; D010453:Peptide Synthases / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011099:Polyglutamic Acid / Q000138:chemical synthesis* / Q000302:isolation & purification; D011624:Pteroylpolyglutamic Acids / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000302:isolation & purification; D013696:Temperature; D014169:Transformation, Bacterial
D005493:Folic Acid Antagonists; D011624:Pteroylpolyglutamic Acids; D011099:Polyglutamic Acid; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase