Technical Comments

Proofreading by Isoleucyl-transfer RNA Synthetase

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Science  11 Dec 1998:
Vol. 282, Issue 5396, pp. 1955
DOI: 10.1126/science.282.5396.1955b

In their report “Enzyme structure with two catalytic sites for double-sieve selection of substrate,” Osamu Nurekiet al. (1) describe the crystal structure of an interesting enzyme: isoleucyl–transfer RNA synthetase (IleRS). The so-called CP1 editing domain of this molecule (1, p. 580) is capable of hydrolyzing misacylated transfer RNA to ensure fidelity in messenger RNA translation. Although this is an excellent study, figure 4 and the discussion of it on the same page (p. 581) do not seem to build a reasonable argument. The figure is a “stereoview of the IleRS editing site as a ball-and-stick representation.”

First, the hydrolysis mechanism cannot be similar to any serine proteases, as implied by Nureki et al. This is because the so-called Thr233-His319-Asn237 triad of amino acid residues appears to be at least 9 Å distant from the bound valine, and Thr233 is not essential for catalysis (according to data presented in the report).

Second, the catalytic role of amino acids Thr230 and Asn237 is unfounded. Asn237, being about 15 Å distant, and connected through a non-essential Thr233 (the white dotted lines in figure 4 in the report), cannot play a direct role in catalysis. Even the role of Thr230 appears unlikely. The Thr230 hydroxyl is either 5.0 Å (as in the text) or 5.5 Å (as in figure 4) distant from a carboxyl oxygen of bound valine (another carboxyl oxygen appears to be missing), and either distance is too far to permit this hydroxyl to be the catalytic nucleophile. It could possibly play this role if the Thr230 site chain could be rotated 180° and one more oxygen could be added to the bound valine, or if some unreported residues (for example His384) could come into the action, or if the bound valine did not reflect the actual substrate binding mode.

Finally, the comparison to ValRS is also inconsistent. While the Trp232 to Glu mutation is probably well served, the His319-Lys and Asn237-Asp mutations may not be meaningful. This comparison would put Thr230 into an arginine in ValRS, an unlikely scenario to replace the proposed catalytic role of Thr230.


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