Glutamine phosphoribosylpyrophosphate amidotransferase from cloned Escherichia coli purF. NH2-terminal amino acid sequence, identification of the glutamine site, and trace metal analysis.
Glutamine 5-phosphoribosylamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was purified in large amounts from an Escherichia coli strain harboring a purF hybrid plasmid. Purified E. coli amidophosphoribosyltransferase lacks iron as well as other trace metals as determined by x-ray fluorescence spectrometry. The NH2-terminal amino acid sequence of the enzyme was determined and is in agreement with that deduced from the DNA sequence. [6-14C] Diazo-5-oxo-norleucine (DON), an active site-directed affinity analog of glutamine, selectively inactivated the glutamine-dependent amidophosphoribosyltransferase. Inactivation was accompanied by incorporation of 1 eq of [6-14C]DON per enzyme subunit. A 10-residue cyanogen bromide peptide labeled by [6-14C]DON was isolated and sequenced. The NH2-terminal cysteine of amidophosphoribosyltransferase was determined to be the residue alkylated by [6-14C]DON. These results establish that the NH2-terminal cysteine is the active site residue required for the glutamine amide transfer function of the enzyme. The experiments reported in this and the preceding article (Tso, J. Y., Zalkin, H., van Cleemput, M., Yanofsky, C., and Smith, J. M. (1982) 257, 3525-3531) demonstrate the application of affinity labeling, rapid peptide purification by high pressure liquid chromatography, and nucleotide sequence determination of a structural gene to localize an amino acid residue, peptide fragment, or functional domain in a long protein chain.
D000582:Amidophosphoribosyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D005796:Genes; D005973:Glutamine / Q000378:metabolism; D010430:Pentosyltransferases / Q000235:genetics*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D014131:Trace Elements / Q000032:analysis*
D010446:Peptide Fragments; D014131:Trace Elements; D005973:Glutamine; D010430:Pentosyltransferases; D000582:Amidophosphoribosyltransferase