摘要
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.
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<sup>-1</sup> min<sup>-1</sup> 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. [<sup>14</sup>C]NEM--labeled LeuRS was subjected to tryptic digestion, andpeptides were separated and sequenced. 179 Cys<sup>*</sup>-Asp-Thr-Leu182 was identified as the major[<sup>14</sup>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.
基金
Supported by the National Natural Science Foundation of China and the President's Research Foundation of Academia Sinica.
