Amino Acid Selectivity in the Aminoacylation of Coenzyme A and RNA Minihelices by Aminoacyl-tRNA Synthetases*

Coenzyme A (CoA-SH), a cofactor in carboxyl group activation reactions, carries out a function in nonribosomal peptide synthesis that is analogous to the function of tRNA in ribosomal protein synthesis. The amino acid selectivity in the synthesis of aminoacyl-thioesters by nonribosomal peptide synthetases is relaxed, whereas the amino acid selectivity in the synthesis of aminoacyl-tRNA by aminoacyl-tRNA synthetases is restricted. Here I show that isoleucyl-tRNA synthetase aminoacylates CoA-SH with valine, leucine, threonine, alanine, and serine in addition to isoleucine. Valyl-tRNA synthetase catalyzes aminoacylations of CoA-SH with valine, threonine, alanine, serine, and isoleucine. Lysyl-tRNA synthetase aminoacylates CoA-SH with lysine, leucine, threonine, alanine, valine, and isoleucine. Thus, isoleucyl-, valyl-, and lysyl-tRNA synthetases behave as aminoacyl-S-CoA synthetases with relaxed amino acid selectivity. In contrast, RNA minihelices comprised of the acceptor-TψC helix of tRNAIle or tRNAVal were aminoacylated by cognate synthetases selectively with isoleucine or valine, respectively. These and other data support a hypothesis that the present day aminoacyl-tRNA synthetases originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis, functionally similar to the present day nonribosomal peptide synthetases.

editing mechanism of these AARSs destroys the homocysteinyl-AMP intermediate with the formation of the thioester homocysteine thiolactone (3)(4)(5)(6); this prevents utilization of homocysteine in protein synthesis (1,2,11). Thioester bond formation during editing of homocysteine is facilitated by a subsite that binds the side chain thiol of homocysteine. Such thiol binding subsites exist in the synthetic/editing active sites of MetRS (7), IleRS (8), and LysRS (6). Similar thiol binding sites also exist in active sites of AARSs that apparently do not need editing function, such as ArgRS (9), AspRS (6), and SerRS (6). The thiol binding site confers on each of these six AARSs the ability to catalyze aminoacylation of thiols, including CoA-SH, and to promote peptide bond formation (6 -9). CoA-SH binds to an AARS at a site, separate from the tRNA and ATP binding sites, that includes the thiol-binding subsite (10). Since the CoA-SHbinding site provides a thiol-binding subsite that may potentially be utilized for editing of homocysteine, it has been suggested that the editing site for homocysteine has evolved from the thiol-binding portion of the CoA-SH site (10).
IleRS activates not only isoleucine but also homocysteine, cysteine, valine, threonine, alanine, serine, and leucine (1,2). The editing mechanism of IleRS destroys the noncognate intermediates and assures that only isoleucine is transferred to tRNA Ile . In contrast, RNA minihelix comprised of the acceptor-TC helix of tRNA Ile has been reported to be aminoacylated with valine, as well as with isoleucine, by IleRS (13). However, the amino acid selectivity of the CoA-SH aminoacylation reaction is not known. Here, I show that IleRS and ValRS exhibit relaxed amino acid selectivity in the CoA-SH aminoacylation reaction and transfer noncognate amino acids to the thiol group of CoA-SH. It is also shown that the amino acid selectivity of IleRS and ValRS in the aminoacylation of RNA minihelix Ile and RNA minihelix Val , respectively, is much greater than in the aminoacylation of S-CoA-SH. In addition, it is shown that LysRS catalyzes aminoacylation of CoA-SH, but not of RNA minihelix Lys , with lysine and other amino acids.
Radiolabeled  14 C-labeled amino acid. Aliquots of the aminoacylation mixtures were analyzed by polyacrylamide gel electrophoresis at pH 5.5 (11,12). Formation of aminoacylated RNA minihelices could not be monitored by precipitation with 5% trichloroacetic acid due to incorporation of 14  TLC Analyses-Products of the reactions of CoA-SH with activated amino acids were analyzed by TLC on PEI-cellulose (from Merck or Sigma) using 0.8 M LiCl as a solvent. Reaction mixtures were also analyzed by TLC on cellulose plates (Eastman Kodak Co.) using butanol:acetic acid:water (4:1:1, v/v) as a solvent. TLC plates were autoradiographed for 17 h using Kodak BioMax MR-1 film.

IleRS, ValRS, and LysRS Acylate CoA-SH with Noncognate
Amino Acids-IleRS was incubated with 14 C-labeled amino acids, ATP, and CoA-SH. Aliquots of the reaction mixtures were analyzed using two TLC systems. The PEI-cellulose TLC system separates amino acids, which migrate in the upper quarter of the TLC plate, from aminoacyl-S-CoA thioesters, which migrate in the middle of the TLC plate (10). In the cellulose TLC system aminoacyl-S-CoA thioesters stay at the origin, whereas free amino acids migrate with R F values of 0.2-0.8 (10). New 14 C-labeled compounds, migrating in the middle of a PEI-cellulose TLC plate (Fig. 1A) and staying at the origin of a cellulose TLC plate ( courses of aminoacyl-S-CoA formation catalyzed by IleRS and ValRS are shown in Fig. 3. IleRS-dependent aminoacylation of CoA-SH with the cognate isoleucine was about 2-, 6-, and 20-fold faster than the aminoacylation with noncognate valine, leucine, or alanine, respectively (Fig. 3A). However, ValRS-dependent aminoacylation of CoA-SH with the cognate valine was about 3-and 10-fold slower than the aminoacylation with noncognate alanine and threonine, respectively, and about as fast as the aminoacylation of CoA-SH with isoleucine (Fig. 3B).
Isoleucine was the most efficient amino acid substrate in the IleRS-dependent CoA-SH aminoacylation reaction (Table I). Catalytic efficiencies (k cat /K m ) for CoA-SH aminoacylation with valine, leucine, alanine, and threonine were 10-, 57-, 340-, and 370-fold lower, respectively, than the catalytic efficiency for the aminoacylation with isoleucine. The discrimination between amino acids was mostly due to variations in K m values.
In ValRS-dependent CoA-SH aminoacylation reactions, valine was the most efficient substrate (Table I). Catalytic efficiencies for threonine, alanine, serine, and isoleucine were 2.4-, 6.6-, 19.4-, and 28-fold lower, respectively, than the catalytic efficiency for the aminoacylation with valine. Like with IleRS, the discrimination between amino acids was mostly due to variations in K m values. However, with the exception of isoleucine, k cat values for noncognate amino acids were up to 9-fold higher than for the cognate valine. Overall, the amino acid selectivity of ValRS is somewhat lower that the selectivity of IleRS in the CoA-SH aminoacylation reaction.
As shown in Table II, LysRS catalyzed aminoacylation of CoA-SH with several amino acids. The highest rates of CoA-SH aminoacylation were observed with threonine and leucine (both 2.7 M/h). Rates of CoA-SH aminoacylation with lysine, alanine, valine, and isoleucine were 1.4-, 1.6-, 4-, and 11-fold slower, respectively, than rates observed with threonine and leucine. With all tested amino acids, the formation of AA-S-CoA was abolished by the addition of unlabeled lysine, confirming that LysRS is responsible for their formation.
AARSs catalyze the synthesis of cognate aminoacyl-S-CoAs using either AARS-bound aminoacyl adenylate or aminoacyl-tRNA as a donor, suggesting the existence of a CoA-SH binding site on an AARS (10). The formation of noncognate aminoacyl-S-CoAs is unlikely to be due to a reaction of CoA-SH with noncognate AA-AMP dissociated from the enzyme. For example, upper limits of the dissociation rate constants for Ile-AMP and Val-AMP from corresponding complexes with IleRS are 0.12 min Ϫ1 and 0.16 min Ϫ1 (3), too low to account for the rate constants for IleRS-dependent formation of Ile-S-CoA (1.1 min Ϫ1 ) and Val-S-CoA (1.7 min Ϫ1 ) ( Table I).
Although IleRS catalyzed the transfer of noncognate amino acids to CoA-SH (Table I), there was no transfer of the noncognate amino acids to tRNA Ile (not shown). ValRS catalyzed the transfer of noncognate amino acids to CoA-SH (Table I), but, under similar experimental conditions, no transfer of noncognate amino acids to tRNA Val could be detected (not shown). The lack of stable transfer of noncognate amino acids to native tRNA Ile and tRNA Val has been well documented by other investigators (1,2). LysRS-dependent transfer of noncognate amino acids to tRNA Lys is Ͼ10,000-fold slower than the transfer of lysine to tRNA Lys (11). However, for most noncognate amino acids the rates of the transfer to CoA-SH are similar to the rate of lysine transfer to CoA-SH catalyzed by LysRS (Table  II). Thus, for each examined AARS, the amino acid selectivity in the CoA-SH aminoacylation reaction is much lower than the selectivity in the tRNA aminoacylation reaction.
Aminoacylations of RNA Minihelices-RNA minihelices comprised of the acceptor-TC helix of tRNA Ile (13) and tRNA Val (14) are substrates for aminoacylations by cognate AARSs, albeit some 10 6 -fold less efficient than native tRNA. Aminoacylations of minihelix Ile and minihelix Val were carried out under conditions identical to those used for the aminoacylation of CoA-SH. When incorporation of radiolabeled amino acids into trichloroacetic acid precipitable material was measured, similar low degrees of incorporation were observed for all radiolabeled amino acids tested (not shown), consistent with published data (13). However, trichloroacetic acid precipitation could also monitor nonspecific incorporation of radiolabeled amino acids into protein (15,16), particularly at high concentrations of AARSs used in the minihelix aminoacylation mixtures. To circumvent this problem, reaction mixtures were analyzed by electrophoresis on acid polyacrylamide gels which specifically monitors aminoacyl-RNA (12). As shown in Fig. 4 1-6) and IleRS-dependent aminoacylation of RNA minihelix Ile (lanes 7-12) with [ 14 C]serine (lanes 1 and 7), [ 14 C]alanine (lanes 2 and  8), (lanes 3 and 9), [ 14 C]leucine (lanes 4 and 10),  Remarkably, amino acid selectivities of IleRS and ValRS in the RNA minihelix aminoacylation reaction (Fig. 4) are much higher than the corresponding selectivities in the CoA-SH aminoacylation ( Figs. 1 and 2). For example, the preferred amino acid substrates for RNA minihelix aminoacylation are the cognate amino acids, isoleucine for IleRS (Fig. 4, lane 9) and valine for ValRS (Fig. 4, lane 6). In contrast, ValRS-dependent aminoacylation of CoA-SH with threonine was 10-fold faster than with valine; IleRS-dependent aminoacylations of CoA-SH with valine and leucine occurred at rates similar to the rate observed with isoleucine (Table I).
Nordin and Schimmel (13) reported that IleRS aminoacylates RNA minihelix Ile with valine at about a third of the rate observed with isoleucine. However, data presented here indicate that the incorporation of valine into RNA minihelix Ile cannot be greater that 1% of the incorporation of isoleucine, as directly observed on acid gels (compare lane 12 with lane 9 in Fig. 4). A possible explanation for an apparently 30-fold higher incorporation of valine observed by Nordin and Schimmel is that they used a trichloroacetic acid precipitation method which could have been compromised by amino acid incorporation into protein (15,16), also observed in the present work. DISCUSSION This paper demonstrates that 1) IleRS, ValRS, and LysRS exhibit relaxed amino acid specificity in the aminoacylation of CoA-SH and promote synthesis of noncognate aminoacyl-S-CoA thioesters; 2) the RNA minihelix aminoacylation reactions catalyzed by IleRS and ValRS are much less efficient, but exhibit more stringent amino acid selectivities, than the CoA-SH aminoacylation reactions; LysRS does not catalyze aminoacylation of RNA minihelix Lys .
The aminoacylation of CoA-SH catalyzed by AARSs (10) is reminiscent of the transient formation of aminoacyl-S-pantetheine thioesters by nonribosomal peptide synthetases (18 -20) and, despite the lack of similarities in primary structures (19), suggests a functional link between these two amino acid activating systems (10). The present paper, demonstrating the ability of AARSs to utilize noncognate amino acids in the CoA-SH aminoacylation reaction points to another functional similarity: the relaxed amino acid selectivity in the thioester bond formation reaction is characteristic of both AARSs and nonribosomal peptide synthetases (20). The relaxed amino acid selectivity may be indicative of more primitive aminoacylation systems.
The ability of AARSs to catalyze aminoacylation of CoA-SH could be a vestige from a "thioester world" (17) in which ancestral AARSs may have provided (and possibly utilized) aminoacyl thioesters for nonribosomal peptide synthesis, similar to the present day thiotemplated RNA-independent synthesis of microbial peptide antibiotics (18 -20). Remnants of the thioester world are still present: thioesters are immensely important in biological systems (17). For example, CoA-SH and pantetheine participate in carboxyl group activation reactions, with the exception of coded protein synthesis (17)(18)(19)(20). The nonribosomal peptide synthesis systems utilize as a co-factor phosphopantetheine, whose thiol group serves as an acceptor for amino acids (18, 19).
The thioester-dependent mechanism of peptide synthesis may have preceded the RNA-dependent mechanism in the development of life (17). The ability of the present day AARSs to aminoacylate RNA minihelices was suggested to be an evolutionary vestige from a stage of the development of the aminoacylation function in the RNA world (21). Because it is likely that relaxed amino acid selectivities were a feature of primitive aminoacylation systems, the present findings that AARSs transfer some noncognate amino acids to CoA-SH, but not to RNA-minihelices (e.g. threonine, alanine, and serine by IleRS or isoleucine, alanine, and serine by ValRS) is consistent with the notion that the aminoacylation of thioesters may have originated before the aminoacylation of RNA (17). That structurally diverse class I (e.g. IleRS, ValRS, MetRS) and class II (LysRS, AspRS, SerRS) synthetases have the ability to catalyze the aminoacylation of CoA-SH points to the possible ancient origin of this reaction. After the appearance of aminoacyl-RNA at later stages of evolution, the thioester-dependent and RNAdependent peptide synthesizing systems may have been developing in parallel. Before the advent of coded ribosomal protein synthesis, ancestral AARSs may have facilitated the formation of both aminoacyl-thioesters and aminoacyl-RNAs for noncoded peptide assembly. Ancestral AARSs themselves may have been involved in the thiol-dependent assembly of peptides. A vestige of this peptide forming ability exists in the present day AARSs, which can promote the synthesis of peptides in vitro (6 -10).