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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 ~ (-1) min ~ (-1) respectively. Chemical stoichiometryshowed that only one sulfhydryl group of LeuRS was essential for both activities. protected the active sulfhydryl group from modification, suggesting that the modified sulfhydryl group is located in or near the active site region responsible for amino-acid activation. [~ (14) C] NEM-labeled LeuRS was subjected to tryptic digestion, and peptides 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 observation that the region for Leu- AMP formation was located at the N - terminal part of LeuRS.