Partial purification and properties of an enzyme from Escherichia coli that catalyzes the conversion of glutamic acid and 10-formyltetrahydropteroylglutamic acid to 10-formyltetrahydropterol-gamma-glutamyglutamic acid.
An enzyme that catalyzes the conversion of L-glutamic acid and 10-formyl-H4folic acid (also known as 10-formyl-H4pteroylglutamic acid) to 10-formyl-H4pteroyl-gamma-glutamylglutamic acid has been purified by 74-fold from extracts of Escherichia coli. ATP, Mg-2+, and a monovalent cation (K+ or NH-4, but not Na+) are required for the enzyme to function. Radioactive and bioautographic analyses revealed the formation of a single product. This product was identified as 10-formyl-H-4pteroyl-gamma-glutamylglutamic acid from its spectral characteristics, its ability to be used effectively as a growth faster for Lactobacillus casei 7469, and from radioactive analysis that indicated the incorporation into the product of 1 mol glutamate/mol of 10-formyl-H-4pteroylglutamic acid utilized. The enzyme functions optimally at pH 9.0-9.8 and at 50 degrees. Its molecular weight is estimated at 42,000-43,000. The Km values are 180 muM for L-glutamic acid and less than 2 muM for (-) 10-formyl-H-4pteroylglutamic acid. The only other naturally occurring folate compounds with significant activity as substrate are H-4pteroylglutamic acid and 5,10-methylene-H-4pteroylglutamic acid; however, these compounds are not used as effectively (K-m values are 10-12 mu-M) as 10-formyl-H-4pteroylglutamic acid.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000645:Ammonium Sulfate; D002414:Cations, Monovalent; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D005563:Formic Acid Esters / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D002955:Leucovorin / Q000031:analogs & derivatives; D008274:Magnesium / Q000494:pharmacology; D010453:Peptide Synthases / Q000302:isolation & purification*; D013763:Tetrahydrofolates / Q000378:metabolism*
D002414:Cations, Monovalent; D005563:Formic Acid Esters; D005971:Glutamates; D013763:Tetrahydrofolates; C010161:10-formyltetrahydropteroylglutamic acid; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; D008274:Magnesium; D002955:Leucovorin; D000645:Ammonium Sulfate