Substrate specificity and catalysis by the editing active site of Alanyl-tRNA synthetase from Escherichia coli

Abstract

Aminoacyl-tRNA synthetases (ARSs) enhance the fidelity of protein synthesis through multiple mechanisms, including hydrolysis of the adenylate and cleavage of misacylated tRNA. Alanyl-tRNA synthetase (AlaRS) limits misacylation with glycine and serine by use of a dedicated editing domain, and a mutation in this activity has been genetically linked to a mouse model of a progressive neurodegenerative disease. Using the free-standing Pyrococcus horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNA(Ala) and Ala-tRNA(Ala) as substrates, the deacylation activities of the wild type and five different Escherichia coli AlaRS editing site substitution mutants were characterized. The wild-type AlaRS editing domain deacylated Ser-tRNA(Ala) with a k(cat)/K(M) of 6.6 × 10(5) M(-1) s(-1), equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation but only 12.2-fold greater than the rate with Ala-tRNA(Ala). While the E664A and T567G substitutions only minimally decreased k(cat)/K(M,) Q584H, I667E, and C666A AlaRS were more compromised in activity, with decreases in k(cat)/K(M) in the range of 6-, 6.6-, and 15-fold. C666A AlaRS was 1.7-fold more active on Ala-tRNA(Ala) relative to Ser-tRNA(Ala), providing the only example of a true reversal of substrate specificity and highlighting a potential role of the coordinated zinc in editing substrate specificity. Along with the potentially serious physiological consequences of serine misincorporation, the relatively modest specificity of the AlaRS editing domain may provide a rationale for the widespread phylogenetic distribution of AlaX free-standing editing domains, thereby contributing a further mechanism to lower concentrations of misacylated tRNA(Ala).

Figure 1. Mutagenesis of the AlaRS editing active site

The active site of P. horikoshii AlaX complexed with zinc (orange sphere) and serine (space-filling model) is shown. Side chains adjacent to the serine are labeled in black for P. horikoshii AlaX, while their inferred equivalents in E. coli are indicated in blue. Structural overlays of the AlaX with P. horikoshii AlaRS and A. fulgidis AlaRS, along with sequence alignment of P. horikoshii AlaRS, E. coli AlaRS, and H. sapiens AlaRS, were used to identify comparable E. coli AlaRS residues at the editing active site (also see Ref 37).

Figure 2. Editing of seryl-tRNA^Ala with AlaRS WT and editing mutants

Measurements of editing initial rates with AlaRS WT, T567G, and E664A versus [seryl-tRNA^Ala] were fit to the Michaelis-Menten equation, whereas data obtained with AlaRS Q584H, C666A and I677E were fit by linear regression. The resulting k_cat, K_M, and/or k_cat/K_M parameters are reported in Table 1. The rates shown represent an average of 2–4 independent measurements. Error bars, when shown represent the standard of at least three independent measurements. When no errors bars are shown, each rate represents the average of at two independent experiments, with a variance of no more than 25%.

Figure 3. Deacylation of alanyl-tRNA^Ala with the AlaRS WT and editing mutants

The data for each protein were plotted as in Figure 2, and fit by linear regression as described in “Experimental Procedures.”

Figure 4. Uncatalyzed deacylation of alanyl-tRNA^Ala

Fraction of alanyl-tRNA^Ala remaining (circles) is plotted as a function of time, and the data are fit to the first order equation (shown as a line).

Figure 5. Model of seryl-adenosine in the editing active site

A structural overlay of the P. horikoshii AlaX (blue backbone) and E. coli ThrRS (green backbone) editing active sites is used to show the post transfer editing substrate analog serine 3-aminoadenosine in the active site of AlaX. The zinc atom, oriented towards the β-hydroxyl of serine, is shown as an orange sphere. The AlaX side chains identifiers are indicated in black type, while the corresponding E. coli AlaRS residues are indicated in blue.