## 3 Results
### In Vitro Characterization of the AlaRS C666A Suppressors
As previously described, the AlaRS R561C variant was unable to form mis-servylated tRNA\({}^{\text{Ala}}\) product in vitro, suggesting that an additional conformational change may have occurred in this enzyme leading to greater fidelity during initial serine activation that no longer requires proofreading activity in the editing domain. This hypothesis is further supported by results from the in vivo reporter experiment, which did not reveal Ala to Ser mistranslation in the MG1655 AlaRS R561C mutant background (Figure 3). A secondary measure for determining aaRS proofreading activity is through the monitoring of ATP futile cycling. As has been previously described, the first step in aa-tRNA formation is the activation of free amino acid with ATP to form an aminoacyl-adenylate. During standard cognate aa-tRNA reactions, one molecule of ATP is activated per one aa-tRNA formed, ultimately reaching equilibrium when all tRNA substrates are aminoacylated. Alternatively, when aaRS proofreading is occurring, the mis-activated substrate will be hydrolyzed and can subsequently be re-activated, leading to iterative rounds of ATP consumption. Observations from monitoring ATP cycling suggest that AlaRS R561S was the most proofreading-proficient AlaRS variant as ATP consumption levels surpassed those of the wild-type enzyme (Figure 5G). Furthermore, AlaRS R561S displayed increased rates of ATP consumption when incubated with cognate alanine, consistent with observations that R561S leads to cognate Ala-tRNA\({}^{\text{Ala}}\) deacylation (Figure 5H). While the AlaRS D551Y enzyme displayed the highest rate of Ser-tRNA\({}^{\text{Ala}}\) deacylation in trans, similar levels of ATP consumption were observed when compared to wild-type, suggesting an imbalance in the rate of deacylation and re-activation. Finally, enzymes containing the R561C variant had the lowest rate of ATP consumption, which is consistent with deacylation studies suggesting that these enzymes are unable to proofread Ser-tRNA\({}^{\text{Ala}}\). To further characterize the AlaRS editing suppressors, amino acid activation was monitored using a pyrophosphate (PPi) exchange. This assay enables the determination of amino acid activation kinetics by measuring the accumulation of (\({}^{32}\)P)-ATP in a reaction containing excess (\({}^{32}\)P)-PPi. PPi exchange not only facilitates the determination of aminoacyl-adenylate kinetics, it can also be used to highlight changes in enzyme specificities for different substrates, such as cognate alanine or non-cognate serine. Observations from the pyrophosphate exchange assay indicate that the single AlaRS R561C variant shows increased specificity against serine activation (Table 1). This is consistent with previous observations where the AlaRS R561C variant was unable to hydrolyze Ser-tRNA\({}^{\text{Ala}}\) in trans (Figure 5C) while also being unable to form Ser-tRNA\({}^{\text{Ala}}\) (Figure 5B). The AlaRS suppressor C666A R561C also shows an increased specificity against serine activation as compared to AlaRS C666A (Table 1). This increased serine discrimination by the R561C suppressor may explain the \(\sim\)50% reduction in Ser-tRNA\({}^{\text{Ala}}\) formation (Figure 5B). In contrast, the AlaRS C666A R561S mutant shows similar specificity against serine activation as compared to AlaRS C666A (Table 1). This indicates that the R561S mutation has no effect on amino activation but instead increases overall proofreading as evidenced by the deacylation studies (Figure 5C,D) and ATP futile cycling (Figure 5G,H). Finally, the single AlaRS D551Y variant and the suppressor AlaRS C666A D551Y show reduced specificity against serine activation as seen in Table 1. However, the AlaRS D551Y variant showed the highest rate of deacylation for Ser-tRNA\({}^{\text{Ala}}\) (Figure 5E). Thus, the D551Y substitution is likely productive for Ser-tRNA\({}^{\text{Ala}}\) proofreading but not for preventing serine misactivation.