Purification of glutamine tRNA synthetase from Saccharomyces cerevisiae. A monomeric aminoacyl-tRNA synthetase with a large and dispensable NH2-terminal domain.
Glutamine tRNA synthetase from Saccharomyces cerevisiae has been purified to homogeneity and shown to be a monomer of 91 kDa. The size of the polypeptide agrees with that predicted from the previously reported translated DNA sequence. Mild tryptic digestion removes an amino-terminal domain and releases a fragment of 65 kDa which begins at Ser205. This tryptic fragment is similar in size and sequence to Escherichia coli glutamine tRNA synthetase and shows a modest increase from the full-length yeast enzyme in the Km values for glutamine and ATP and no difference in the kcat for aminoacylation or the Km for tRNA. Thus, the removal of the NH2-terminal domain appears to indirectly affect the ATP- and glutamine-binding sites in the nucleotide-binding fold domain to which the NH2-terminal domain is fused. A monoclonal antibody directed against the NH2-terminal domain of the full-length enzyme has little effect upon the aminoacylation activity. Therefore, over 200 amino acids of the NH2 terminus of the full-length enzyme form a domain that operationally has only a modest influence on the catalytic core of the protein. These studies reinforce the concept that eukaryotic synthetases have quasi-independent domains not found in their prokaryotic counterparts which may confer a function distinct from aminoacylation.
Ludmerer
S W
SW
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Wright
D J
DJ
Schimmel
P
P
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D014357:Trypsin; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase