Identification of amino acid residues modified by two ATP analogs in Bacillus subtilis glutamine synthetase.
Bacillus subtilis glutamine synthetase was modified by two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-triphosphate (8-N3-ATP), each one containing either Mg2+ or Mn2+. The FSBA labeled peptide was monitored by measuring the characteristic absorbance of the 4-carboxybenzenesulfonyl (CBS) part at 243 nm. The 8-N3ATP photolabeled peptide could also be monitored by measuring its absorption at 310 nm. A single CBS-labeled tryptic peptide was obtained, spanning residues 89-91 from the N-terminal of the subunit polypeptide chain, and sequence analysis by Edman degradation revealed that CBS-arginine was at position 91. The amino acids photolabeled by 8-N3ATP at the ATP-binding site in B. subtilis GS were His-186, His-187, and Trp-424. These results suggested that these four amino acids constitute an ATP-binding active site located at the interface between two subunits. The region surrounding Trp-424, which varies among different prokaryotic enzymes, was considered to be involved in a catalytic or regulatory role in B. subtilis GS. Since the same amino acids were labeled when B. subtilis GS was modified with FSBA or 8-N3ATP in the presence of Mn2+ or Mg2+, no conformational difference between B. subtilis GS binding Mn(2+)-ATP and that binding Mg(2+)-ATP was detected by affinity labeling with ATP analogs.
Laboratory of Biochemistry, College of Science, Rikkyo (St. Paul's) University, Tokyo.
E
E
Tanaka
K
K
Kimura
D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000187:drug effects; D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites / Q000187:drug effects; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000187:drug effects; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D008345:Manganese; C017040:8-azidoadenosine 5'-triphosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium; D000241:Adenosine