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. Author manuscript; available in PMC: 2022 Nov 21.

Published in final edited form as: Biochemistry. 2017 Jul 7;56(28):3619–3631. doi: 10.1021/acs.biochem.7b00114

Figure 3. — Amino acid activation and the rate of histidine transfer is unaffected by the Y454S mutation even in the presence of tRNA^His. Amino acid activation (WT: panel A; Y454S: panel B) was monitored by mixing 5 μM monomer HARS enzyme (WT: filled circles; Y454S: filled triangles) with 100 μM ATP and saturating histidine. tRNA^His (25 μM) addition (empty circles and empty triangles, insets: note 0.5 s time scale) increased the rate of amino acid activation, which was determined by linear fit within the first turnover. The rate constant for histidine transfer k_trans (WT: panel C; Y454S: panel D) was determined by single turnover with 2 μM enzyme, preformed with histidyl-adenylate, and mixed with 100 nM tRNA^His. k_trans was determined by fit to a single exponential. The number of experiments is reported in Tables 2 and 3.

Figure 3. — Amino acid activation and the rate of histidine transfer is unaffected by the Y454S mutation even in the presence of tRNA^His. Amino acid activation (WT: panel A; Y454S: panel B) was monitored by mixing 5 μM monomer HARS enzyme (WT: filled circles; Y454S: filled triangles) with 100 μM ATP and saturating histidine. tRNA^His (25 μM) addition (empty circles and empty triangles, insets: note 0.5 s time scale) increased the rate of amino acid activation, which was determined by linear fit within the first turnover. The rate constant for histidine transfer k_trans (WT: panel C; Y454S: panel D) was determined by single turnover with 2 μM enzyme, preformed with histidyl-adenylate, and mixed with 100 nM tRNA^His. k_trans was determined by fit to a single exponential. The number of experiments is reported in Tables 2 and 3.