## 3 Results
### AlaRS R561S Has Elevated Proofreading Activity  
In contrast to observations studying the effect of the AlaRS R561C on aaRS discrimination, the AlaRS R561S variant appears to be hyper-active in AlaRS proofreading, as indicated by the increased hydrolytic activity. The AlaRS R561S C666A variant is able to mis-sersylate tRNA\({}^{\text{Ala}}\) which is consistent with the observation of Ala to Ser mistranslation in vivo (Figure 3). Furthermore, this enzyme shows no changes in the discrimination of serine during amino acid activation, as compared to AlaRS C666A (Table 1). However, the levels of Ser-tRNA\({}^{\text{Ala}}\) do appear to be slightly reduced compared to the AlaRS C666A single mutant (Figure 5A). The elevated proofreading activity is more obvious when monitoring the deacylation activity directly in editing assays. The R561S variant has elevated rates of Ser-tRNA\({}^{\text{Ala}}\) deacylation compared to wild-type AlaRS (Figure 5E), and the R561S C666A enzyme has elevated proofreading activity compared to editing-defective AlaRS C666A variant (Figure 5F). Consistent with these observations is the elevated levels of ATP consumption in enzymes containing the R561S substitution. The single AlaRS R561S variant had the highest rate of ATP consumption and the R561S C666A variant had elevated ATP consumption compared to the C666A enzyme (Figure 5G). Interestingly, the R561S enzyme had elevated levels of deacylation (Figure 5D) and ATP consumption for cognate Ala-tRNA\({}^{\text{Ala}}\) substrates (Figure 5H). This observation provides the first evidence of a hyper-active proofreading enzyme. Furthermore, the contribution of AlaRS R561S together with the editing-defective C666A substitution may lead to an overall reduction of Ser-tRNA\({}^{\text{Ala}}\) in the cell.  
\begin{table}
\begin{tabular}{c c c c c c} \hline  & \multicolumn{3}{c}{**Ala**} & & \\ \cline{2-6}  & \(K_{\text{sat}}\) & \(k_{\text{cat}}\) & \(k_{\text{cat}}\)/\(K_{\text{sat}}\) & **Ser \({}^{\text{a}}\)**(\(k_{\text{cat}}\)/\(K_{\text{sat}}\))** & **Specificity \({}^{\text{b}}\)**(Ala/Ser)** \\  & \(\mu\)**M** & **min\({}^{-1}\)** & **min\({}^{-1}\)/\(\mu\)M** & **min\({}^{-1}\)/\(\mu\)M** & \\ \hline \(E\). coli/ AlaRS & \(320\pm 150\) & \(1246\pm 105\) & \(3.91\) & \(4.7\times 10^{-3}\pm 1.5\times 10^{-4}\) & \(832\) \\ AlaRS C666A & \(263\pm 115\) & \(1010\pm 380\) & \(3.83\) & \(7.3\times 10^{-3}\pm 5.5\times 10^{-4}\) & \(525\) \\ AlaRS D551Y & \(260\pm 76\) & \(715\pm 220\) & \(2.76\) & \(6.9\times 10^{-3}\pm 2.5\times 10^{-4}\) & \(400\) \\ AlaRS D551Y C666A & \(217\pm 100\) & \(1096\pm 265\) & \(5.06\) & \(10.4\times 10^{-3}\pm 14\times 10^{-4}\) & \(490\) \\ AlaRS R561C & \(203\pm 68\) & \(1306\pm 345\) & \(6.45\) & \(6.7\times 10^{-3}\pm 13\times 10^{-4}\) & \(963\) \\ AlaRS R561C & \(109\pm 30\) & \(819\pm 88\) & \(4.29\) & \(5.5\times 10^{-3}\pm 19\times 10^{-4}\) & \(780\) \\ AlaRS R561S & \(180\pm 15\) & \(1216\pm 125\) & \(6.70\) & \(8.2\times 10^{-3}\pm 19\times 10^{-4}\) & \(817\) \\ AlaRS R561S C666A & \(177\pm 37\) & \(791\pm 150\) & \(4.46\) & \(7.3\times 10^{-3}\pm 5\times 10^{-4}\) & \(611\) \\ \hline \multicolumn{6}{l}{a \(K_{\text{cat}}\)/\(K_{\text{sat}}\) was estimated using subsaturating Ser concertrations from the slope of the equation, \(\text{V}=K_{\text{cat}}\)[E]S/\(K_{\text{sat}}\); b} \\ \multicolumn{6}{l}{Measulted in \(K_{\text{cat}}\)/\(K_{\text{sat}}\).} \\ \end{tabular}
\end{table}
Table 1: Steady state activation kinetics of AlaRS C666A suppressors. Steady state kinetic parameters of the AlaRS C666A suppressors for the activation of alanine and serine were determined using pyrophosphate exchange.