Aminoacyl adenylates and

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.

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. 

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-... 

Jhe synthesis of proteins, as characterized by the in vitro incorporation of amino acids into the protein component of cytoplasmic ribonu-cleoprotein, is known to require the nonparticulate portion of the cytoplasm, ATP (adenosine triphosphate) and GTP (guanosine triphosphate) (15, 23). The initial reactions involve the carboxyl activation of amino acids in the presence of amino acid-activating enzymes (aminoacyl sRNA synthetases) and ATP, to form enzyme-bound aminoacyl adenylates and the enzymatic transfer of the aminoacyl moiety from aminoacyl adenylates to soluble ribonucleic acid (sRNA) which results in the formation of specific RNA-amino acid complexes—see, for example, reviews by Hoagland (12) and Berg (1). The subsequent steps in pro-... 

FIGURE 12.6 The aminoacyl-tRNA synthetase reaction, (a) The overall reaction. Everpresent pyrophosphatases in cells quickly hydrolyze the PPj produced in the aminoacyl-tRNA synthetase reaction, rendering aminoacyl-tRNA synthesis thermodynamically favorable and essentially irreversible, (b) The overall reaction commonly proceeds in two steps (i) formation of an aminoacyl-adenylate and (ii) transfer of the activated amino acid moiety of the mixed anhydride to either the 2 -OH (class I aminoacyl-tRNA synthetases) or 3 -OH (class II aminoacyl-tRNA synthetases) of the ribose on the terminal adenylic acid at the 3 -OH terminus common to all tRNAs. Those aminoacyl-tRNAs formed as 2 -OH esters undergo a transesterification that moves the aminoacyl group to the 3 -OH of tRNA Only the 3 -esters are substrates for protein synthesis. 

Suppose that a particular aminoacyl-tRNA synthetase has a f0% error rate in the formation of aminoacyl-adenylates and a 99% success rate in the hydrolysis of incorrect aminoacyl-adenylates. What percentage of the tRNAs produced by this aminoacyl-tRNA synthetase will be faulty ... 

Figure 13.15 Reaction sequences for aminoacyl-tRNA S5mthesis. Synthesis of aminoacyl-tRNA catalyzed aminoacyl-tRNA syntheses (aRS) occurs in two steps, formation of enzyme-associated (activated) aminoacyl adenylate and transfer of the activated aminoacyl group to form esoteric linkage with either the 2 -OH (class I aRS) or 3 -OH (class II aRS) of the ribose on the terminal 3 -CCA of tRNA. The transesterification converts 2 -0-aminoacyl-tRNA to 3 -0-aminoacyl-tRNA which is the substrate for protein synthesis...

Because the nature of the bond fonnalion process is different in PKs and NRPs (Claisen condensations vs. peptide bond formalion), NRPSs necessarily utilize different domains. Specifically, the three essential domains of the NRPS are an adenylation (A), peptide carrier protein (PCP), and condensation (C) domain. NRP biosynthesis begins with activation of the amino add through adenylation of the carboxylic acid group by the A domain, a process facilitated by the presence of Mg +. In the adenylation reaction, ATP reacts with the selected amino acid to form the activated phosphoester aminoacyl adenylate and pyrophosphate (Figure 4.10a). The A domain is also a key point of selectivity during NRP biosynthesis as it is responsible for the selection of the particular amino acid to be activated. 

The hydrolytic stability of branched RNA where the branch-point is a phospho-triester unit revealed that when the triester was embedded within the oligonucleotide it was stable to base-mediated hydrolysis by more than an order of magnitude compared to the simple trinucleotide unit. 5 -Aminoalkyl phosphate nucleotides have been synthesised as analogues of aminoacyl adenylates, and salicyl phospho-diesters of adenylates as potential inhibitors of Mycobacterium tuberculosis. ... 

Figure 21 Two step activation of an amino acid substrate in non-ribosomal biosynthesis as aminoacyl adenylate and enzyme thioester. Both steps are catalysed by the same enzyme which catalyses amino acyladenylate formation (top) and covalently hinds its amino acid substrate as thioester (bottom)...

In some cases, mutation can lead to enhanced catalytic ability of the enzyme. Results for the mutation Thr-51 to Pro-51 (Wilkinson et al 1984) have been mentioned previously. The results for this and for the mutation Thr-51 to Ala-51 (Fersht e/ al., 1985) are also shown in Table 18. These mutations and that of Thr-51 to Cys-51 have been studied in some detail (Ho and Fersht, 1986). In each case it is found that the transition state is stabilized for formation of tyrosine adenylate from tyrosine and ATP within the enzyme the mutant Thr-51 to Pro-51 increases the rate coefficient for the reaction by a factor of 20. However, the enzyme-bound tyrosine adenylate is also stabilized by the mutation and this results in a reduced rate of reaction of tyrosine adenylate with tRNA (48), the second step in the process catalysed by tyrosine tRNA synthetase. Overall, therefore, the mutants are poorer catalysts for the formation of aminoacyl tRNA. The enzyme from E. coli has the residue Pro-51 whereas Thr-51 is present in the enzyme from B. stearothermophilus. The enzyme from E. coli is more active than the latter enzyme in both the formation of tyrosine adenylate and in the aminoacyla-tion of tRNA (Jones et al., 1986b). It is therefore suggested (Ho and Fersht, 1986) that the enzyme from E. coli with Pro-51 must additionally have evolved ways of stabilizing the transition state for formation of tyrosine adenylate without the concomitant stabilization of tyrosine adenylate and reduction in the rate of aminoacylation of tRNA found for the Pro-51 mutant. 

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. ... 

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... 

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