Aminoacyl adenylate

Figure 4.30 Structures of an aminoacyl-adenylate and of an aminoacyl-tRNA.

Figure 12 Aminoacyl-adenylates and stable bioisosteres. Ad, adenosine R, amino acid side chain.

Aminoacyl adenylates (296), which are formed from protein amino acids and ATP, act as acylating agents towards t-RNAs, acylating their terminal 3 -hydroxy groups. These charged tRNAs are then used in protein synthesis. Little is known about the reactivity of aminoacyl adenylates (296), and studies are now reported of a model compound, alanyl ethyl phosphate (297). As expected, hydrolysis in both acid and base involves attack at the C=0 group of (297) with departure of ethyl phosphate. Metal ions (Cu +, Zn +) were found to act as catalysts of the hydrolysis. 

First, the amino acid is bound by the enzyme and reacts there with ATP to form diphosphate and an energy-rich mixed acid anhydride (aminoacyl adenylate). In the second step, the 3 -OH group (in other ligases it is the 2 -OH group) of the terminal ribose residue of the tRNA takes over the amino acid residue from the aminoacyl adenylate. In aminoacyl tRNAs, the carboxyl group of the amino acid is therefore esterified with the ribose residue of the terminal adenosine of the sequence. ..CCA-3. ... 

This reaction occurs in two steps in the enzyme s active site. In step (Fig. 27-14) an enzyme-bound intermediate, aminoacyl adenylate (aminoacyl-AMP), forms when the carboxyl group of the amino acid reacts with the a-phosphoryl group of ATP to form an anhydride linkage, with displacement of pyrophosphate. In the sec-... 

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... 

The aminoacyl-tRNA synthetases join amino acids to their appropriate tRNA molecules for protein synthesis. They have the very important task of selecting both a specific amino acid and a specific tRNA and joining them. The enzymes differ in size and other properties. However, they all appear to function by a common basic chemistry that makes use of cleavage of ATP at Pa (Eq. 12-48) via an intermediate aminoacyl adenylate and that is outlined also in Eq. 17-36. These enzymes are discussed in Chapter 29. ... 

The soluble enzyme system responsible for its synthesis contains a large 280-kDa protein that not only activates the amino acids as aminoacyl adenylates and transfers them to thiol groups of 4 -phosphopantetheine groups covalently attached to the enzyme but also serves as a template for joining the amino acids in proper sequence.211-214 Four amino acids—proline, valine, ornithine (Om), and leucine—are all bound. 

Mechanisms of reaction. Activation of an amino acid occurs by a direct in-line nucleophilic displacement by a carboxylate oxygen atom of the amino acid on the a phosphorus atom of MgATP to form the aminoacyl adenylate (Eq. 29-1, step a). For yeast phenylalanyl-tRNA synthetases the preferred form of MgATP appears to be the P,y-bidentate (A screw sense) complex (p. 643).250 This is followed by a second nucleophilic displacement, this one on the C = 0 group of the aminoacyl adenylate by the -OH group of the tRNA (Eq. 29-1, step b Fig. 29-9C). A conformational change in the protein may be required to permit dissociation of the product, the aminoacyl-tRNA. In the complex of a class I synthetase with aminoacyl... 

In the two classes of synthetase the tRNAs approach the enzyme in a mirror-symmetric fashion. The 2 -OH of the terminal ribose is positioned to attack the carbonyl of the aminoacyl adenylate in class I enzymes, while the 3 -OH is positional for the attack in class II enzymes.207... 

An example of the application of these equations is found in Chapter 7, section D. The aminoacyl-tRNA synthetase that specifically esterifies the tRNA molecule that accepts valine, tRNAVal, corrects the error when it mistakenly forms an aminoacyl adenylate with threonine by the following scheme ... 

There is no doubt that the enzyme-bound aminoacyl adenylate is formed in the absence of tRNA. It may be isolated by chromatography and the free aminoacyl adenylate obtained by precipitation of the enzyme with acid.47 48 Furthermore, the isolated complex will transfer its amino acid to tRNA. The crystal structure of the tyrosyl-tRNA synthetase bound to tyrosyl adenylate has been solved (Chapter 15, section B). 

Despite this, it seemed at one stage that not all the evidence was consistent 5 with the aminoacyl adenylate pathway. An alternative mechanism appeared possible in the presence of tRNA, perhaps an aminoacyl adenylate was not formed, and the reaction occurred instead by the simultaneous reaction of the tRNA, the... 

When IRS, [14C]Ile, tRNA, and ATP are mixed in the quenched-flow apparatus (Figure 7.6), the initial rate of charging of tRNA extrapolates back through the origin without any indication of a burst of charging. The burst of pyrophosphate release is due to the formation of the aminoacyl adenylate before the transfer of the amino acid to tRNA. 

Figure 13.4 The double sieve analogy for the editing mechanism of the isoleucyl-tRNA synthetase. The active site for the formation of the aminoacyl adenylate can exclude amino acids that are larger than isoleucine but not those that are smaller. On the other hand, a hydrolytic site that is just large enough to bind valine can exclude isoleucine while accepting valine and all the smaller amino acids. (In some enzymes, the hydrolytic site offers specific chemical interactions that enable it to bind isosteres of the correct amino acid as well as smaller amino acids.)...

In the absence of tRNA, the enzymes will, with a few exceptions, activate amino acids to the attack of nucleophiles, and ATP to the attack of pyrophosphate.43 6 This is done by forming a tightly bound complex with the aminoacyl adenylate, the mixed anhydride of the amino acid, and AMP. (The chemistry of activation is discussed in Chapter 2, section D2c.)... 

The aminoacyl adenylate pathway is proved very simply from three quenched-flow experiments by using the three criteria for proof the intermediate is isolated it is formed fast enough and it reacts fast enough to be on the reaction pathway.50 The following is found for the isoleucyl-tRNA synthetase (IRS) ... 

When IRS, isoleucine, tRNA, and [y-32P]ATP (labeled in the terminal phosphate) are mixed in the pulsed quenched-flow apparatus (Figure 7.5), there is a burst of release of labeled pyrophosphate before the steady state rate of aminoacylation of tRNA is reached. This means either that the aminoacyl adenylate is formed before the aminoacylation of tRNA, thus proving the... 

There are two high-energy intermediates on the reaction pathway that could be edited by hydrolysis the enzyme-bound aminoacyl adenylate and the aminoacyl-tRNA. A mechanistic study must distinguish between the two. A pathway involving the mischarged tRNA involves the formation of a covalent intermediate—the aminoacylated tRNA—so the three rules of proof may be considered (Chapter 7, section Al). These criteria have been rigorously applied to the rejection of threo-... 

Aminoacyl adenylates have long been known to be high energy compounds, but their free energies of hydrolysis had not been accurately measured. This was accomplished for tyrosyl adenylate using the Haldane approach (Chapter 3, section H) and mutants of the tyrosyl-tRNA synthetase. The equilibrium constant for the formation of tyrosyl adenylate in solution (Absolution) = [Tyr-AMP] [PPi]/ [Tyr] [ATP]) is related to the rate and equilibrium constants for the enzymatic reaction illustrated in Figure 15.21 by equation 15.8. 

Nucleoside phosphoacyl (36) Aminoacyl adenylate Competitive inhibition +... 

The aminoacyl adenylates react rapidly with amino acids to yield peptides39 , under physiological conditions in aqueous solution at room temperature. At pHs higher than 7, the maximum is attained at pH 10 where the extent of the polymerization of peptides from alanyl adenylate is about 60 % 40). The aminoacyl adenylate in aqueous solution undergoes an intra- or inter-molecular rearrangement with the formation of 2 (3 )-aminoacyl ester of adenosine 40). 

Basic proteins, histones, increase the rate of peptide formation from aminoacyl 5 -adenylates in neutral solution 41 . The acceleration is about threefold in the initial phase of the reaction. A mixture of histone and polyribonucleotide also increases the rate of peptide formation 41>. Histone might offer local basic surroundings to the reacting aminoacyl adenylate molecules 41 . 

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