Leucyl-tRNA synthetase (LeuRS, EC 6.1.1.4) from E. coli underwent limitedproteolysis by trypsin which cut off 6K peptide and converted the intact LeuRS into a 96K fragment. The truncated enzyme retained the PPi exchan...Leucyl-tRNA synthetase (LeuRS, EC 6.1.1.4) from E. coli underwent limitedproteolysis by trypsin which cut off 6K peptide and converted the intact LeuRS into a 96K fragment. The truncated enzyme retained the PPi exchange activity with the same kinetic parameters as those of native LeuRS but lost the tRNA^(Leu)charging, binding and other tRNA^(Leu)-related activities. N-terminus analysis showed that the 6K peptide was located at the C-terminus of LeuRS. This small part played a crucial role in tRNA^(Leu) binding. Our results suggest that the two activities, PPi exchange and tRNA charging are independent of each other and correspond to different structural regions of LeuRS. The C-terminal region might be the tRNA^(Leu)binding site of LeuRS.展开更多
The chemical modification of the sulfhydryl groups of E. coli Leucyl--tRNA synthetase(LeuRS) by DTNB, NEM and IAA resulted in a time-dependent loss of both amino-acid acti-vation and aminoacylation activities in paral...The chemical modification of the sulfhydryl groups of E. coli Leucyl--tRNA synthetase(LeuRS) by DTNB, NEM and IAA resulted in a time-dependent loss of both amino-acid acti-vation and aminoacylation activities in parallel. The second-order reaction constants of DTNB,NEM and IAA were 1700, 150 and 0.46 mol/L^(-1) min^(-1) respectively. Chemical stoichiometryshowed that only one sulfhydryl group of LeuRS was essential for both activities. Substratesleucine and Leu-AMP protected the active sulfhydryl group from modification, suggestingthat the modified sulfhydryl group is located in or near the active site region responsiblefor amino-acid activation. [^(14)C]NEM--labeled LeuRS was subjected to tryptic digestion, andpeptides were separated and sequenced. 179 Cys~*-Asp-Thr-Leu182 was identified as the major[^(14)C]NEM-labeled site in LeuRS. This result is consistent with the previous observationthat the region for Leu--AMP formation was located at the N--terminal part of LeuRS.展开更多
基金Project supported by the National Natural Science Foundation of China. Part of the results was presented at the 4th Congress of FAOB (Federation of Asian and Oceanian Biochemists) in 1986.
文摘Leucyl-tRNA synthetase (LeuRS, EC 6.1.1.4) from E. coli underwent limitedproteolysis by trypsin which cut off 6K peptide and converted the intact LeuRS into a 96K fragment. The truncated enzyme retained the PPi exchange activity with the same kinetic parameters as those of native LeuRS but lost the tRNA^(Leu)charging, binding and other tRNA^(Leu)-related activities. N-terminus analysis showed that the 6K peptide was located at the C-terminus of LeuRS. This small part played a crucial role in tRNA^(Leu) binding. Our results suggest that the two activities, PPi exchange and tRNA charging are independent of each other and correspond to different structural regions of LeuRS. The C-terminal region might be the tRNA^(Leu)binding site of LeuRS.
基金Supported by the National Natural Science Foundation of China and the President's Research Foundation of Academia Sinica.
文摘The chemical modification of the sulfhydryl groups of E. coli Leucyl--tRNA synthetase(LeuRS) by DTNB, NEM and IAA resulted in a time-dependent loss of both amino-acid acti-vation and aminoacylation activities in parallel. The second-order reaction constants of DTNB,NEM and IAA were 1700, 150 and 0.46 mol/L^(-1) min^(-1) respectively. Chemical stoichiometryshowed that only one sulfhydryl group of LeuRS was essential for both activities. Substratesleucine and Leu-AMP protected the active sulfhydryl group from modification, suggestingthat the modified sulfhydryl group is located in or near the active site region responsiblefor amino-acid activation. [^(14)C]NEM--labeled LeuRS was subjected to tryptic digestion, andpeptides were separated and sequenced. 179 Cys~*-Asp-Thr-Leu182 was identified as the major[^(14)C]NEM-labeled site in LeuRS. This result is consistent with the previous observationthat the region for Leu--AMP formation was located at the N--terminal part of LeuRS.
