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The mechanismof this step is somewhat different for the twoclasses of aminoacyl-tRNA synthetases (see Table26-7). For class I enzymes, the aminoacyl group istransferred initially to the 2'-hydroxyl group of the3'-terminal adenylate residue, then moved to the 3'hydroxyl by a transesterification reaction. For classII enzymes, the aminoacyl group is transferred directly to the 3' hydroxyl of the terminal adenylate,as shown.C—O—P—O—CH2NH 3 O0~OHOH5'-Aminoacyl adenylate(aminoacyl-AMP)class IIaminoacyl-tRNAsynthetasesclass Iammoac\ 1-tRXAs\nthetases3' end of tRNAR—CI+OOHC—O—P—O— AdenosineC—O—P—O— AdenosineII UI+NH 3 ONH 3 OO"Aminoacyl-AMPO"Aminoacyl-AMPTable 26-7 Twoclasses ofaminoacyl-tRNAsynthetases*HO—^C—C—RHC—C—R6 + NH 36+NH3TransesterificationAminoacyl-tRNAClass IClass IIArgAlaCysAsnGinAspGluGlylieHisLeuLysMetTVpTVrPheValThrProSer"" Classification applies to allorganisms for which tRNAsynthetases have been analyzed and is based on protein structural distinctionsand on the mechanistic distinction outlined in Figure26-18Chapter 26 Protein Metabolism913Aminoacyl-tRNA Synthetases Attach the CorrectAmino Acids to Their tRNAsIn the first stage of protein synthesis, which takes place in the cytosol,the 20 different amino acids are esterified to their correspondingtRNAs by aminoacyl-tRNA synthetases, each of which is specific forone amino acid and one or more corresponding tRNA.
In most organisms there is generally one aminoacyl-tRNA synthetase for each aminoacid. As noted earlier, for amino acids that have two or more corresponding tRNAs the same aminoacyl-tRNA synthetase usuallyaminoacylates all of them. In E. coli, the only exception to this rule islysine, for which there are two aminoacyl-tRNA synthetases. There isonly one tRNALys in E. coli, and the biological rationale for the presence of two Lys-tRNA synthetases is unclear. Nearly all the aminoacyltRNA synthetases of E. coli have been isolated; all have been sequenced (either the protein itself or its gene), and a number have beencrystallized.
They have been divided into two classes (Table 26-7)based on distinctions in primary and tertiary structure and on differences in reaction mechanism, as detailed below. The overall reactioncatalyzed by these enzymes isAmino acid + tRNA + ATPaminoacyl-tRNA + AMP +The activation reaction occurs in two separate steps in the enzymeactive site.
In the first step, an enzyme-bound intermediate, aminoacyladenylate (aminoacyl-AMP) is formed by reaction of ATP and theamino acid at the active site (Fig. 26-18). In this reaction, the carboxylgroup of the amino acid is bound in anhydride linkage with the 5'phosphate group of the AMP, with displacement of pyrophosphate.In the second step the aminoacyl group is transferred fromenzyme-bound aminoacyl-AMP to its corresponding specific tRNA. Asshown in Figure 26-18, the course of this second step depends upon theclass to which the enzyme belongs (Table 26-7).
The reason for themechanistic distinction between the two enzyme classes is unknown.The resulting ester linkage between the amino acid and the tRNA (Fig.26-19) has a high standard free energy of hydrolysis (AGO/ =- 2 9 kJ/mol). The pyrophosphate formed in the activation reactionundergoes hydrolysis to phosphate by inorganic pyrophosphatase.Thus two high-energy phosphate bonds are ultimately expended foreach amino acid molecule activated, rendering the overall reaction foramino acid activation essentially irreversible:Amino acid + tRNA + ATPMg2aminoacyl-tRNA + AMP + 2V{AG°' - -29kJ/molSome Aminoacyl-tRNA Synthetases Are Capableof ProofreadingThe aminoacylation of tRNA accomplishes two things: the activation ofan amino acid for peptide bond formation and attachment of the aminoacid to an adapter tRNA that directs its placement within a growingpolypeptide.
As we will see, the identity of the amino acid attached to atRNA is not checked on the ribosome. Attaching the correct amino acidto each tRNA is therefore essential to the fidelity of protein synthesisas a whole.3' end of tRNAHIO—C6C-R+NH3AminoacylgroupFigure 26-19 General structure of aminoacyltRNAs. The aminoacyl group is esterified to the 3'position of the terminal adenylate residue. Theester linkage that both activates the amino acidand joins it to the tRNA is shaded red.Part IV Information Pathways914ValineIsoleucineThe potential for any enzyme to discriminate between two different substrates is limited by the available binding energy that can bederived from enzyme-substrate interactions (Chapter 8).
Discrimination between two similar amino acid substrates has been studied indetail in the case of Ile-tRNAIle synthetase, which faces the molecularproblem that valine differs from isoleucine only by one methylene(CH2) group. For this enzyme, activation of isoleucine (to form IleAMP) is favored over valine by a factor of 200, in the range expectedgiven the potential contribution of binding energy from a methylenegroup. However, valine is incorporated into proteins in positions normally occupied by isoleucine at a frequency of only about 1 in 3,000.The difference is brought about by a separate proofreading function of Ile-tRNA synthetase; this function is also present in some otheraminoacyl-tRNA synthetases.
All aminoacyl-AMPs produced by IletRNA synthetase are checked in a second active site on the same enzyme, and incorrect ones are hydrolyzed. This proofreading activityreflects a general principle already seen in the discussion of proofreading by DNA polymerases (p. 822). If available binding interactions involving different groups on two substrates do not provide for a sufficient discrimination between the two on the enzyme, then thisavailable binding energy must be used twice (or more) in separatesteps requiring discrimination.
Forcing the system through two successive "filters" rather than one increases the potential fidelity by apower of 2. In the case of Ile-tRNA synthetase, the first filter is theinitial amino acid binding and activation to aminoacyl-AMP. The second filter is the separate active site, which catalyzes deacylation ofincorrect aminoacyl-AMPs.
The aminoacyl-AMP intermediates remainbound to the enzyme. When tRNAIle binds to the enzyme, the presenceof Ile-AMP leads to aminoacylation of the tRNA. If Val-AMP is presenton the enzyme instead, it is hydrolyzed to valine and AMP and thetRNA is not aminoacylated. Because the R group of valine is slightlysmaller than that of isoleucine, the Val-AMP fits the hydrolytic (proofreading) site of the Ile-tRNA synthetase, but Ile-AMP does not.In addition to proofreading after formation of the aminoacyl-AMPintermediate, most aminoacyl-tRNA synthetases are also capable ofhydrolyzing the ester linkage between amino acids and tRNAs in aminoacyl-tRNAs.
This hydrolysis is greatly accelerated for incorrectlycharged tRNAs, providing yet a third filter to enhance the fidelity ofthe overall process. In contrast, in a few aminoacyl-tRNA synthetasesthat activate amino acids that have no close structural relatives, littleor no proofreading occurs; in these cases the active site can sufficientlydiscriminate between the proper substrate amino acid and incorrectamino acids.The overall error rate of protein synthesis (~1 mistake per 104amino acids incorporated) is not nearly as low as for DNA replication,perhaps because a mistake in a protein is erased by destroying theprotein and is not passed on to future generations.
This degree of fidelity is sufficient to ensure that most proteins contain no mistakes andthat the large amount of energy required to synthesize a protein israrely wasted.The Interaction between Aminoacyl-tRNA Synthetase andtRNA Constitutes a "Second Genetic Code"An individual aminoacyl-tRNA synthetase must be specific not only fora single amino acid but for a certain tRNA as well. DiscriminatingChapter 26 Protein Metabolismamong several dozen tRNAs is just as important for the overall fidelityof protein biosynthesis as is distinguishing among amino acids. Theinteraction between aminoacyl-tRNA synthetases and tRNAs has beenreferred to as the "second genetic code," to reflect its critical role inmaintaining the accuracy of protein synthesis.
The "coding" rules areapparently more complex than those in the "first" code.Figure 26-20 summarizes what is known about the nucleotidesinvolved in recognition by some or all aminoacyl-tRNA synthetases.Some nucleotides are conserved in all tRNAs and therefore cannot beused for discrimination. Nucleotide positions that are involved in discrimination by the aminoacyl-tRNA synthetases have been identifiedby the fact that changes at those nucleotides alter the enzyme's substrate specificity. These interactions seem to be concentrated in theamino acid arm and the anticodon arm, but are also located in manyother parts of the molecule.
The conformation of the tRNA (as opposedto its sequence) can also be important in recognition.Some aminoacyl-tRNA synthetases recognize the tRNA anticodonitself. Changing the anticodon of one tRNAVal from UAC to CAU makesthis tRNA an excellent substrate for Met-tRNA synthetase. The ValtRNA synthetase will similarly recognize a modified tRNAMet in whichthe anticodon has been changed to UAC. Recognition by aminoacyltRNA synthetases of other tRNAs (about half of them, including thosefor alanine and serine) is affected little or not at all by changes at theanticodon. In some cases ten or more specific nucleotides are involvedin recognition of a tRNA by its specific aminoacyl-tRNA synthetase(Fig. 26-20). In contrast, across a range of organisms from bacteria tohumans the primary determinant for tRNA recognition by the AlatRNA synthetases is a single G = U base pair in the amino acid arm oftRNA^ a (Fig.
26-21a). A short RNA with as few as seven base pairsarranged in a simple hairpin minihelix is efficiently aminoacylated bythe Ala-tRNA synthetase as long as the RNA contains this criticalG=U (Fig. 26-21b).915Amino acidarm51T(//C armDHU arm•(Extra armAnticodonarmAnticodonFigure 26-20 Known positions in tRNAs recognized by aminoacyl-tRNA synthetases. Positionsin blue are the same in all tRNAs and thereforecannot be used to discriminate one from another.Other positions are known recognition points forone (red) or more (green) tRNA synthetases. Structural features other than sequence are importantfor recognition by some tRNA synthetases.3'76\ Deleted-t o y nucleotides(a)(b)Figure 26-21 (a) The tRNA^3 structural elementsrecognized by the Ala-tRNA synthetase are unusually simple. A single G=U base pair (red) is theonly element needed for specific binding andaminoacylation.
(b) A short synthetic RNA minihelix, which has the critical G=U base pair butlacks most of the remaining tRNA structure, isspecifically aminoacylated with alanine almostas efficiently as the complete tRNAAla.Figure 26-22 Structure of Gln-tRNA synthetase(white) bound to its cognate tRNAGln (green andred) and ATP.
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