Technical Comments

Proofreading by Isoleucyl-Transfer RNA Synthetase: Response

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Science  22 Jan 1999:
Vol. 283, Issue 5401, pp. 459
DOI: 10.1126/science.283.5401.459a

In our report of 24 April 1998, we described the structure of isoleucyl–transfer RNA (tRNA) synthetase and noted that it has two catalytic sites: one for aminoacylation and one for editing of a misactivated substrate (1). This enzyme, like other isoleucyl-tRNA synthetases, misactivates valine to form valyl adenylate. On addition of tRNAIle, the valyl adenylate is either hydrolyzed to valine and adenosine 5′-monophosphate [(AMP), which eliminates the misactivated valine], or the valyl moiety is transferred onto tRNAIle to form Val-tRNAIle; an esterase activity then rapidly removes Val from tRNAIle(2). Thus, the tRNAIle-dependent editing activity eliminates misactivated valine, whether in the form of valyl-AMP or Val-tRNAIle.

In our report (1), we identified the editing site by solving the structure of a cocrystal with valine. In contrast to isoleucine, which bound only at the active site, valine was bound to two sites—the active site and a second site in an insertion known as CP1 (3). We also presented results of a mutational analysis of some of the residues found near the bound valine within CP1. Collectively, these results suggested some of the residues that are and are not important for editing activity.

In his technical comment of 11 December 1998, Xiayang Qiu criticizes this analysis along specific lines (4). He first focuses on a particular catalytic triad of residues at the active site for editing. We indeed mentioned in passing that five residues in the site which binds L-valine (Thr228, Thr230, Thr233, Asn237, and His319) are located in close proximity in a way that “is reminiscent of the catalytic triads of hydrolases.” (Thr229 should also have been mentioned. We follow the numbering system of the Thermus thermophilus enzyme, the structure of which we described in this report; most of the mutational analysis was done on theEscherichia coli homolog.) Qiu then states that we must be suggesting a catalytic triad of Thr233-His319- Asn237. But in the report, we specifically state that mutation of Thr233 (and of Thr228) had no effect on the editing response. Thus, we did not suggest that the triad attributed to us by Qiu was of catalytic significance.

Qiu states that the idea that Thr230 and Asn237 have a catalytic role in editing “is unfounded.” This statement is puzzling in that we reported that mutation of either of these residues affects editing. [As a result of a clerical error, Thr229 (which corresponds to Thr242 in the E. coli system) appeared incorrectly as “Thr230” twice in the first paragraph on page 581 of our report (1)]. Qiu does not offer an explanation for this experimental result. Moreover, he states that the threonine hydroxyl group is too far (5 to 5.5 Å) from bound valine to serve as the catalytic nucleophile. Yet we did not suggest that a threonine hydroxyl is the catalytic nucleophile. As we stated, residues that are mutated and thereby affect editing have a catalytic role—meaning that the rate (kcat/Km) for editing is dependent on these residues. The detailed nature of this dependence remains to be determined. As for the catalytic nucleophile, among the possibilities is the tightly bound water (1) at the editing site.

Last, we would not take a “static” view of the editing site, as Qiu seems to do in his comment. The outstanding problem in the field is to determine how misactivated substrates move from the active site to the editing site. Most likely, a significant conformational change is involved. Thus, the distances quoted by Qiu between side chain groups in the protein and bound valine are relevant only to the crystal form that we studied. We imagine that some conformational shifts take place during an editing reaction. For that reason, we regarded all residues with functional groups that are within 15 Å or so of the bound valine to be of potential importance. (Another consideration for us was that valine is just a fragment of the larger substrates that are edited—that is, valyl adenylate and Val- tRNAIle—so that a radius around the bound valine needs to be considered.) Thus, for example, Thr229 and Asn237could be important in a “static” or rigid editing site through action-at-a distance; alternatively, one or both of these residues could directly play a role in catalysis by virtue of a localized rearrangement that brings either closer to the valyl moiety in the transition state of editing. Regardless of the detailed interpretation, our most recent experiments have confirmed the importance for editing of this region of CP1: for example, the Thr229A mutant enzyme mischarges tRNAIle with valine to yield Val-tRNAIle, a reaction typically not seen with wild-type isoleucyl-tRNA synthetase (5).


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