Covalent modification of substrate-binding sites of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-ADP-glucose-incorporated peptides.
A photoaffinity substrate analogue, 8-azido-ADP-[14C]glucose, reacts specifically and covalently with Escherichia coli ADP-glucose synthetase. The site(s) of reaction of 8-azido-ADP-[14C]glucose with the enzyme was identified by isolation of tryptic peptides containing the labeled analogue by use of high performance liquid chromatography technique and subsequent NH2-terminal sequence analysis of the purified radioactive peptides. One major binding region of the azido analogue is a peptide segment composed of residues 107-114 of the enzyme's polypeptide chain. Lys 108 and Arg 114 become trypsin-resistant sites when the enzyme is photoinactivated by 8-azido-ADP-[14C] glucose, suggesting that the analogue binds at or near the vicinity of these 2 basic amino acid residues. Conformational analysis of this peptide segment (residues 107-114) shows a strong probability of a reverse beta-turn secondary structure, suggesting that this peptide segment is on the enzyme surface. Two minor reaction regions of the enzyme with the analogue were also identified by chemical characterization. One region was composed of residues 162-207. Lys 194 was previously suggested as the activator-binding site by chemical modification studies with pyridoxal phosphate (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). Another minor region where the analogue binds the tryptic peptide composed of residues 380-385 is near the COOH-terminal side of the enzyme. It is postulated that all these peptide segments are juxtaposed in tertiary structure.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D051599:Glucose-1-Phosphate Adenylyltransferase; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D001386:Azides; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D014357:Trypsin; C021622:8-azidoadenosine diphosphate