Aminoacylation, specificity

Figure 23 Differentiation of initiation and elongation epimerization domains by intermodular transfer activity, (a) Intermodular transfer activity assay. Peptidyl or aminoacyl specificity of epimerization domains is monitored by tripeptide formation or dipeptide formation, respectively, (b) Proposed mechanism of aminoacyl epimerization domain and peptidyl epimerization domain in NRPS.42...

The ionizable functional groups of aminoacyl side chains and (where present) of prosthetic groups contribute to catalysis by acting as acids or bases. Acid-base catalysis can be eithet specific ot general. By specific we mean only protons (HjO ) or OH ions. In specific acid or specific base catalysis, the rate of reaction is sensitive to changes in the concenttation of protons but... 

The regions of the tRNA molecule teferred to in Chapter 35 (and illustrated in Figure 35-11) now become important. The thymidine-pseudouridine-cyti-dine (T PC) arm is involved in binding of the amino-acyl-tRNA to the ribosomal surface at the site of protein synthesis. The D arm is one of the sites important for the proper recognition of a given tRNA species by its proper aminoacyl-tRNA synthetase. The acceptor arm, located at the 3 -hydroxyl adenosyl terminal, is the site of attachment of the specific amino acid. 

Fig. 5.1 Simplified model representation of the activation of an amino acid (ASY) at an amino acid-activating enzyme (i.e., an amino acid-specific aminoacyl-tRNA synthetase)...

The information contained in the DNA (i.e., the order of the nucleotides) is first transcribed into RNA. The messenger RNA thus formed interacts with the amino-acid-charged tRNA molecules at specific cell organelles, the ribosomes. The loading of the tRNA with the necessary amino acids is carried out with the help of aminoacyl-tRNA synthetases (see Sect. 5.3.2). Each separate amino acid has its own tRNA species, i.e., there must be at least 20 different tRNA molecules in the cells. The tRNAs contain a nucleotide triplet (the anticodon), which interacts with the codon of the mRNA in a Watson-Crick manner. It is clear from the genetic code that the different amino acids have different numbers of codons thus, serine, leucine and arginine each have 6 codewords, while methionine and tryptophan are defined by only one single nucleotide triplet. 

Thompson, Damien Plateau, Pierre Simonson, Thomas, Free-energy simulations and experiments reveal long-range electrostatic interactions and substrate-assisted specificity in an aminoacyl-tRNA synthetase., ChemBioChem Feb 2006, 7, 337-344. 

To determine the efficiency of aminoacylation of [14C]Phe-tRNA, 5 fil aliquots of the aminoacylation mixture are withdrawn before and after the reaction the samples taken from the reaction mixture at the end of the incubation are spotted onto 3-MM paper discs (Schleicher Schuell) and processed by the cold TCA precipitation method, while the sample taken before the reaction is spotted on a paper disc pretreated empty by the same cold TCA procedure. Determination of the radioactivity present on these filters by liquid scintillation counting allows one to calculate the aminoacylation efficiency of the reaction (which, for phenylalanine, should be >2% of total tRNA). The specific activity of the [14C] Phe-tRNA can be determined after one-step purification of Phe-tRNA by BD cellulose chromatography (Gillam et al., 1968), followed by determination of the radioactivity and of the A260. 

Although aminoacyl-tRNA synthetases are necessary for protein synthesis in all tissues, their importance in chemical carcinogenesis is difficult to assess. Mutation induction by this pathway has been studied extensively (123), yet metabolic activation in a carcinogen-target tissue has not been demonstrated. The only exception is hepatic prolyl-tRNA synthetase activation of N-hydroxy-Trp-P-2 however, hepatic O-acetylation of this substrate also occurs to an appreciable extent (12). Further investigations involving the use of specific enzyme inhibitors would be helpful in addressing this problem. 

Activation of individual amino acids occurs in the synthesis of aminoacyl tRNA. This process bums two ATP equivalents (forms pyrophosphate and AMP) and connects a specific amino acid to a specific tRNA. 

How, in turn, does the synthetase recognize its specific tRNA From extensive mutagenesis studies, it appears that the aminoacyl-tRNA synthetases recognize particular regions of the tRNA molecule, most often in their anticodon loops and/or in their acceptor stems. 

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