Threonyl-tRNA synthetase

Mehlotate hydrolase and threonyl-tRNA synthetase have been reported to use this reaction scheme. The... 

The zinc site is less well suited to discrimination against serine because this amino acid does have a hydroxyl group that can bind to the zinc. Indeed, with only this mechanism available, threonyl-tRNA synthetase does mistakenly couple serine to threonyl-tRNA at a rate 10 2 to 10 3 times that for threonine. As noted in Section 29. LL this error rate is likely to lead to many translation errors. How is a higher level of specificity achieved ... 

Threonyl-tRNA synthetase can be incubated with tUNA hr that has been covalently linked with serine (Ser-tRNAThr) the tRNA has been "mischarged." The reaction is immediate a rapid hydrolysis of the aminoacyl-tRNA forms serine and free tRNA. In contrast, incubation with correctly charged Thr-tRNAThr results in no reaction. Thus, threonyl-tRNA synthetase contains an additional functional site that hydrolyzes Ser-tRNA hr but not Thr-tHNA hr This editing site provides an opportunity for the synthetase to correct its mistakes and improve its fidelity to less than one mistake in 10. The results of structural and mutagenesis studies revealed that the editing site is more than 20 A from the activation site (Figure 29.9). This site readily accepts and cleaves Ser-tRNAThr but does not cleave Thr-tRNA hr The discrimination of serine from threonine is relatively easy because threonine contains an extra methyl group a site that conforms to the structure of serine will sterically exclude threonine. 

At least one aminoacyl-tRNA synthetase exists for each amino acid. The diverse sizes, subunit composition, and sequences of these enzymes vv ere be vildering for many years. Could it be that essentially all synthetases evolved independently The determination of the three-dimensional structures of several synthetases follo ved by more-refined sequence comparisons revealed that different synthetases are, in fact, related. Specifically, synthetases fall into tvv o classes, termed class I and class II, each of vv hich includes enzymes specific for 10 of the 20 amino acids (Table 29.2). Glutaminyl-tRNA synthetase is a representative of class I. The activation domain for class I has a Rossmann fold (Section 16.1.101. Threonyl-tRNA synthetase (see Figure 29.11) is a representative of class II. The activation domain for class II consists largely of P strands. Intriguingly, synthetases from the tvv o classes bind to different faces of the tRNA molecule (Figure 29.14). The CCA arm of tRNA adopts different conformations to accommodate these interactions the arm is in the helical conformation observed for free tRNA (see Figures 29.5 and 29.6) for class II enzymes and in a hairpin conformation for class I enzymes. These two classes also differ in other ways. 

Figure 29.9. Editiug Site. The results of mutagenesis studies revealed the position of the editing site (shown in green) in threonyl-tRNA synthetase.

Figure 29.11. Threonyl-tRNA Synthetase Complex. The structure of the complex between threonyl-tRNA synthetase and tRNAThr reveals that the synthetase binds to both the acceptor stem and the anticodon loop.

Zinc ion mediated amino acid discrimination by threonyl-tRNA synthetase Nat. Struct. Biol. 7 461-465. 

R. Sankaranarayanan, A.C. Dock-Bregeon, P. Romby, J. Caillet, M. Springer, B. Rees, C. Ehresmann, B. Ehresmann, and D. Moras. 1999. The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the achve site Cell 97 371-381. (PubMed)... 

From Voet Voet, 2004) (b) The amino acid-binding site of threonyl-tRNA synthetase showing the amino acid bound to a zinc atom through its amino and hydroxyl groups (From Berg et ai, 2002. Reproduced with permission from W.H. Freeman and Co.)... 

The structure of the amino acid-binding site of threonyl-tRNA synthetase reveals how valine is avoided (Figure 30.7). 3 he enzyme contains a zinc ion, bound to the enzyme by two histidine residues and one cysteine... 

Sankaranarayanan, R., Dock-Bregeon, A. C., Rees, B., Bovee, M., Caillet, J., Romby, R. Fraiicklyn, C. S., and Moras. D. 2000. Zinc ion niediated amino acid discrimination by threonyl-tRNA synthetase. Nat. Struct. Biol. 7 461—465. 

Active site of threonyl-tRNA synthetase Figure 30.7... 

Coordination of the threonine hydroxyl by an active site Zn in the threonyl-tRNA synthetase allows discrimination between threonine and the isosteric valine (Sankaranarayanan et al., Nat. Struct. Biol. 7[2000] 461-465). Given the similarity of serine and threonine (Ser lacks only the methyl group of Thr), if this is the only mechanism for amino acid discrimination available, threonyl-tRNA synthetase mistakenly couples Ser to threonyl-tRNA at a rate several-fold higher than it does threonine. Since this would lead to unacceptably high error rates in translation, how it is it avoided ... 

Threonyl-tRNA synthetase has a proofreading mechanism. Any Ser-tRNA that is mistakenly formed is hydrolyzed by an editing site 20 A from the activation site. The decision to hydrofyze the aminoacyl-tRNA appears to depend on the size of the amino acid substituent, ff it is smaffer than the correct amino acid, the amino acid fits into the hydrolytic site and is cleaved. If it is the same size as the correct amino acid, it does not fit and is not destroyed. Discrimination between amino acids that are larger than the correct one or are not isoelectronic with it occurs at the aminoacylation step. 

Protein kinase. Ceruloplasmin, Threonyl-tRNA synthetase. Lactate dehydrogenase... 

Sankaranarayanan R, Dock-Bregeon AC, Rees B, Bovee M, Caillet J, Romby P, Francklyn CS, Moras D Zinc ion mediated amino acid discrimination by threonyl-tRNA synthetase. Nat Struct Biol 2000, 7(6) 46l-465. 

Gantt, J.S., Bennett, C.A., and Arfin, S.M., Increased levels of threonyl-tRNA synthetase in a borre-lidin-resistant Chinese hamster ovary cell line, Proc. Natl. Acad. Sci. U.S.A., 78, 5367, 1981. 

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