Glutamine active site of formylglycinamide ribonucleotide amidotransferase. 2. Amino acid sequence of labeled peptides

Publisher Website
Google Scholar
Abstract
The purification and characterization of 2 related peptides making up the glutamine binding site of formylglycinamide ribonucleotide amidotransferase from chicken liver were presented. An amino acid residue(s) involved in binding glutamine to the enzyme was selectively labeled with [14C]iodoacetate. The labeled enzyme was reduced, carboxymethylated and degraded by trypsin to a large radioactive peptide that yielded on acid hydrolysis only cysteine as a radioactive carboxymethylated derivative. The tryptic peptide was further digested with a protease from Streptomyces griseus. Two radioactive fractions (I and II) were obtained by gel filtration on Sephadex G-25. Two 14C-containing peptides were isolated from fraction I by the aid of ion exchange chromatography on AG 1-X2, AG 50W-X2 and DEAE-cellulose. Upon acid hydrolysis both peptides yielded only carboxymethylcysteine (CMCys), cystine, glycine, valine, aspartic acid and glutamic acid. The partial sequences of the amino residues in these peptides, which are located at the glutamine binding site, were established by the dansyl-Edman method. The sequences of amino acids of peptides a and b are Gly-Val-Cys([14C]CM)-Asp-Asx-Cys(CM)-Glx...and Gly-Val-Cys([14C]CM)-Asx-Asx..., respectively. The 2 peptides are undoubtedly derived from the same segment of the original protein.