E292 is important for the aminoacylation activity of Escherichia coli leucyl-tRNA synthetase.
Escherichia coli leucyl-tRNA synthetase (LeuRS) has a large connecting polypeptide (CP1) inserted into its active site. It was demonstrated that the peptide bond between E292-A293 was crucial for the aminoacylation activity of E. coli LeuRS. To investigate the effect of E292 on the function of Escherichia coli LeuRS, E292 was mutated to K, F, S, D, Q and A. These mutations at 292 did not change the specific activity of the amino acid activation reaction. Though the conformational change of these mutants was not detected in CD, their aminoacylation activities were impaired to varying extents. The mutation of E to K decreased the aminoacylation activity to the largest extent. Analysis of the Km values of these mutants for the three substrates showed that the E292 was not involved in the binding of leucine and that all mutants had stronger binding with ATP.
State Key Laboratory of Molecular Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Xing
X
Du
En-Duo
ED
Wang
D000215:Acylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D012356:RNA, Transfer, Leu / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D012356:RNA, Transfer, Leu; D007935:Leucine-tRNA Ligase