title: alpha-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli.
abstract: Folate cofactors in most cells contain polyglutamate side chains, which since the late 1940s have been assumed to be linked via their gamma-COOH groups. We report here an investigation of the structure of the polyglutamate chain attached to the folates of Escherichia coli. Folates were extracted from E. coli grown with [7-14C] p-aminobenzoate and cleaved to p-aminobenzoyl polyglutamates of varying chain lengths (pAB(Glu)n) by the method of Foo et al. (Foo, S. K., Cichowicz, D. J., and Shane, B. (1980) Anal. Biochem. 107, 109-115). The pAB(Glu)n derived from E. coli did not co-chromatograph with chemically synthesized pAB(gamma-Glu)n-Glu on several high performance liquid chromatography (HPLC) systems, except for the triglutamate which did elute with pAB(gamma-Glu)2-Glu. E. coli-derived pAB(Glu)3-8 were purified by HPLC on C18 columns eluted with acetonitrile/trifluoroacetic acid, and the structures were determined through mass spectrometry, chiral amino acid analysis, and peptidase digestion experiments. Molecular weight determinations on the methyl ester derivatives of E. coli-derived pAB(Glu)n by liquid secondary ion mass spectrometry and sequence analysis using collision-activated dissociation on a tandem mass spectrometer confirmed the structures as pAB(Glu)3-8. Chiral HPLC of hydrolyzed and dansylated E. coli-derived materials, on a beta-cyclodextrin column, identified the glutamate as the L-enantiomer. pAB(Glu)n were digested with carboxypeptidase Y, which specifically cleaved glutamates linked at their alpha-carboxyls; E. coli-derived pAB(Glu)4-8 (but not synthetic pAB(gamma-Glu1-6-Glu) were sequentially digested to pAB(gamma-Glu)2-Glu. Thus, in E. coli folylpolyglutamates, glutamate residues 4-8 were each linked to the polyglutamate chain at the alpha-carboxyl of the preceding glutamate.
mesh_terms: D010129:4-Aminobenzoic Acid / Q000378:metabolism*; D062365:Aminobenzoates / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002268:Carboxypeptidases; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000254:growth & development*; D005492:Folic Acid / Q000031:analogs & derivatives*; D005971:Glutamates / Q000032:analysis*; D013058:Mass Spectrometry; D011624:Pteroylpolyglutamic Acids / Q000097:blood / Q000302:isolation & purification*; D013237:Stereoisomerism
chemical_list: D062365:Aminobenzoates; D002250:Carbon Radioisotopes; D005971:Glutamates; D011624:Pteroylpolyglutamic Acids; D005492:Folic Acid; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D010129:4-Aminobenzoic Acid