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. Author manuscript; available in PMC: 2021 Jan 1.
Published in final edited form as: SLAS Discov. 2019 Sep 9;25(1):57–69. doi: 10.1177/2472555219873095

Figure 2.

Figure 2.

Determination of the activity of P. aeruginosa lysyl-tRNA synthetase (LysRS) and the kinetic parameters governing interactions with its three substrates: adenosine triphosphate (ATP), lysine and tRNALys. P. aeruginosa LysRS was titrated into the aminoacylation assay (A) as described in the “Methods and Materials” section in amounts varying from 0.5 to 4 pmol enzyme. Background activity was minimal and was subtracted from values at all concentrations of LysRS. Initial velocities for the interaction of LysRS with both ATP (B) and lysine (C) were determined using the ATP:PPi exchange reaction. The initial velocity for the interaction of LysRS with tRNA was determined using the aminoacylation reaction (D). The concentration of LysRS in the aminoacylation reactions and the exchange reactions was 0.03 μM and 0.1 μM, respectively. Initial velocities were determined, and the data were fit to a Michaelis-Menten steady-state model using XLfit 5.3 (IDBS) to determine KM and Vmax. The kinetic parameters were calculated from these data for the interaction of LysRS with the three substrates (E).