Aminoacyl-tRNA synthetases activation sites

MECHANISM FIGURE 27-14 Aminoacylation of tRNA by aminoacyl-tRNA synthetases. Step is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases (see Table 27-7). For class I enzymes, (2a) the aminoacyl group is transferred initially to the 2 -hydroxyl group of the 3 -terminal A residue, then (3a) to the 3 -hydroxyl group by a transesterification reaction. For class II enzymes, ( the... 

In addition to proofreading after formation of the aminoacyl-AMP intermediate, most aminoacyl-tRNA synthetases can also hydrolyze the ester linkage between amino acids and tRNAs in the aminoacyl-tRNAs. This hydrolysis is greatly accelerated for incorrectly charged tRNAs, providing yet a third filter to enhance the fidelity of the overall process. The few aminoacyl-tRNA synthetases that activate amino acids with no close structural relatives (Cys-tRNA synthetase, for example) demonstrate little or no proofreading activity in these cases, the active site for aminoacylation can sufficiently discriminate between the proper substrate and any incorrect amino acid. 

FIGURE 27-17 Aminoacyl-tRNA synthetases. Both synthetases are complexed with their cognate tRNAs (green stick structures). Bound ATP (red) pinpoints the active site near the end of the aminoacyl arm. 

Amino acids are activated by specific aminoacyl-tRNA synthetases in the cytosol. These enzymes catalyze the formation of aminoacyl-tRNAs, with simultaneous cleavage of ATP to AMP and PPj. The fidelity of protein synthesis depends on the accuracy of this reaction, and some of these enzymes carry out proofreading steps at separate active sites. In bacteria, the initiating aminoacyl-tRNA in all proteins is A-formylmethionyl-tRNAfMet. 

While peptide antibiotics are synthesized according to enzyme-controlled polymerization patterns, both proteins and nucleic acids are made by template mechanisms. Tire sequence of their monomer emits is determined by genetically encoded information. A key reaction in the formation of proteins is the transfer of activated aminoacyl groups to molecules of tRNA (Eq. 17-36). Tire tRNAs act as carriers or adapters as explained in detail in Chapter 29. Each aminoacyl-tRNA synthetase must recognize the correct tRNA and attach the correct amino acid to it. The tRNA then carries the activated amino acid to a ribosome, where it is placed, at the correct moment, in the active site. Peptidyltransferase, using a transacylation reaction, in an insertion mechanism transfers the C terminus of the growing peptide chain onto the amino group of... 

Class II aminoacyl-tRNA synthetases contain a different set of three "signature sequences," two of which form an ATP-binding catalytic domain. The active site structure is built on an antiparallel (3 sheet and is surrounded by two helices (Fig. 29-9). Each class contains subgroups with inserted loops that form other domains. In the following tabulation the reference numbers refer to three-dimensional structural studies. 

Each synthetase module contains three active site domains The A domain catalyzes activation of the amino acid (or hydroxyacid) by formation of an aminoacyl- or hydroxyacyl-adenylate, just as occurs with aminoacyl-tRNA synthetases. However, in three-dimensional structure the A domains do not resemble either of the classes of aminoacyl-tRNA synthetases but are similar to luciferyl adenylate (Eq. 23-46) and acyl-CoA synthetases.11 The T-domain or peptidyl carrier protein domain resembles the acyl carrier domains of fatty acid and polyketide synthetases in containing bound phos-phopantetheine (Fig. 14-1). Its -SH group, like the CCA-terminal ribosyl -OH group of a tRNA, displaces AMP, transferring the activated amino acid or hydroxy acid to the thiol sulfur of phosphopan-tetheine. The C-domain catalyzes condensation (peptidyl transfer). The first or initiation module lacks a C-domain, and the final termination module contains an extra termination domain. The process parallels that outlined in Fig. 21-11.1... 

Specificity is a grossly overworked and often misused word. The most important meaning for the enzymologist refers to an enzyme s discrimination between several substrates competing for an active site for example, the specificity of a particular aminoacyl-tRNA synthetase for a particular amino acid and a particular tRNA in a mixture of all the amino acids and all the tRNAs. This is the... 

The activation and transfer steps for a particular amino acid are catalyzed by the same aminoacyl-tRNA synthetase. Indeed, the aminoacyl-AMP intermediate does not dissociate from the synthetase. Rather, it is tightly bound to the active site of the enzyme by noncovalent interactions. Aminoacyl-AMP is normally a transient intermediate in the synthesis of aminoacyl-tRNA, but it is relatively stable and readily isolated if tRNA is absent from the reaction mixture. 

Aminoacyl-tRNA Synthetases Have Highly Discriminating Amino Acid Activation Sites... 

Most aminoacyl-tRNA synthetases contain editing sites in addition to acylation sites. These complementary pairs of sites fimction as a double sieve to ensure very high fidelity. In general, the acylation site rejects amino acids that are larger than the correct one because there is insufficient room for them, whereas the hydrolytic site cleaves activated species that are smaller than the correct one. 

The answer is c. (Murray, pp 452-467. Scriver, pp 3-45. Sack, pp 1—40. Wilson, pp 101—120.) Two molecules of GTP are used in the formation of each peptide bond on the ribosome. In the elongation cycle, binding of aminoacyl-tRNA delivered by EF-Tu to the A site requires hydrolysis of one GTE Peptide bond formation then occurs. Translocation of the nascent peptide chain on tRNA to the P site requires hydrolysis of a second GTE The activation of amino acids with aminoacyl-tRNA synthetase requires hydrolysis of ATP to AMP plus PP,. 

Describe the two sequential reactions that occur in the active site of aminoacyl-tRNA synthetases. 

It is critical that the correct amino acid is attached to the tRNA. Otherwise, the correct protein will not be synthesized. Fortunately, the synthetases correct then-own mistakes. For example, valine and threonine are approximately the same size— threonine has an OH group in place of a CH3 group of valine. Both amino acids, therefore, can bind at the amino acid binding site of the aminoacyl-tRNA synthetase for valine, and both can then be activated by reacting with ATP to form an acyl adenylate. The aminoacyl-tRNA synthetase for valine has two adjacent catalytic sites, one for... 

---