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  • Title: Lysyl residue 47 is near the subunit ATP-binding site of glutamine synthetase from Escherichia coli.
    Author: Pinkofsky HB, Ginsburg A, Reardon I, Heinrikson RL.
    Journal: J Biol Chem; 1984 Aug 10; 259(15):9616-22. PubMed ID: 6146616.
    Abstract:
    The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) inactivates dodecameric glutamine synthetase from Escherichia coli with concomitant labeling of one site/subunit (Foster, W.B., Griffith, M.J., and Kingdon, H.S. (1981) J. Biol. Chem. 246, 882-886). Cyanogen bromide cleavage of the FSBA-inactivated enzyme in 70% HCOOH produced a large peptide (Mr congruent to 4600) in approximately 75% yield containing N epsilon-4-carboxybenzenesulfonyl (CBS) lysine. The CBS-peptide was purified by high performance liquid chromatography on the basis of its relatively high UV absorbance at 246 nm (delta epsilon congruent to 19,000 M-1 cm-1). The appearance of the labeled peptide coincided with the loss of a peptide (lacking tyrosine and tryptophan) from the unmodified, active enzyme; the CBS-peptide also could be distinguished from cysteine-containing peptides labeled with thionitrobenzoate and from the adenylylated peptide. The identical peptide was labeled when the unadenylylated enzyme was inactivated with FSBA in the absence or presence of Mn2+ or Mg2+ (+/- L-glutamate) or when the enzyme was adenylylated. Conformational differences among these enzyme forms therefore are not detected by affinity labeling of ATP sites. The FSBA-labeled peptide spans residues 9-48 from the N-terminal end of the subunit polypeptide chain, and sequence analysis by automated Edman degradation revealed that CBS-lysine was at position 47. Thus, lysyl residue 47 appears to be near the subunit gamma-phosphate-binding site for ATP in the native glutamine synthetase structure. The primary structure of the CBS-labeled peptide is presented.
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