Ligase aminoacyl

Eigneg E.A. and Loftfield R.B. (1974) Kinetic techniques for the investigation of amino acid tRNA ligases (aminoacyl-tRNA synthetases, amino acid activating enzymes). Methods EnzymoL, 29, 601-619. 

Endonucleases, like pancreatic RNase or takadiesterase (Chapter 3, Section 3.4.2, p. 27) split tRNA to any extent mainly in the loops (10,14,19) (Fig. 8.6). This reaction has been used to split tRNA molecules into halves and quarters, which could be isolated, characterized, and then further degraded. By this means, the nucleotide sequences of many tRNA s have been elucidated (12-14). Furthermore, the reassembly of such fragments has been used to determine which part of the tRNA molecule is necessary for which function. From such studies (19), it was concluded that the recognition by the tRNA ligase (aminoacyl-tRNA-synthetase) takes place in the stem region, and the loops and other helical regions are not involved (Fig. 8.8). 

Ligases forming aminoacyl-tRNA and related compounds... 

From a chemical point of view, the El/ubiquitin thiol ester should be competent to donate ubiquitin to a substrate amino group. In fact, aminoacyl-errzyme thiol esters are used in exactly this way in non-ribosomal polypeptide synthesis, a process that was discovered around the same time as ubiquitin-protein conjugation [5]. In spite of the attractive simplicity of this model, however, biochemical reconstitution studies showed that besides El two additional fractions were required to conjugate ubiquitin to a model substrate. They were called ubiquitin carrier protein (E2) and ubiquitin-protein ligase (E3), respectively, since the respective factors seemed to act sequentially [6]. Interestingly, the E2 factor apparently formed a thiol ester with ubiquitin. Based on these results, Hershko and co-workers proposed the ubiquitin conjugation cascade (Figure 5.1). 

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 enzyme [EC 6.1.1.21], also referred to as histi-dinedRNA ligase, catalyzes the reaction of histidine with tRNA and ATP to produce histidyl-tRNA , AMP, and pyrophosphate. See also Aminoacyl-tRNA Synthetases... 

Nonetheless, there are several unexploited gene families that may provide additional multigene targets for inhibitors, such as the two-component signal transduction systems [32,51], tRNA synthetases [52], RNA polymerase sigma subunits [53,54], and the aminoacyl ligases of peptidoglycan biosynthesis [55],... 

Ligases Ligation of two substrates at the expense of ATP hydrolysis Aminoacyl-tRNA synthetase 29... 

The Lipid I and II building blocks may be further elaborated by many other enzymes that modify the sugars or amino acid chains. Branched peptides are added to the Lipid I and II peptide chains either by enzymes that act in an ATP-dependent fashion similar to the MurC-F ligases [39], or by enzymes that add amino acid residues from aminoacyl tRNA intermediates, such as the S. aureus enzymes FemA, FemB and FemX, which form the pentaglycine bridge (O Fig. 3) [36], and the S. pneumoniae enzymes FemM and FemN, which form an L-Ser-L-Ala or L-Ala-L-Ala dipeptide bridge [34,35]. Lipid II is also the substrate for the sortase enzymes that catalyze the attachment of surface proteins for incorporation into peptidoglycan [40]. 

The aminoacyl-tRNA S5mthetases (amino acid tRNA ligases) join amino acids to their appropriate transfer RNA molecules for protein S5mthesis. They have the very important task of selecting both a specific amino acid and a specific tRNA and joining them according to Eq. These reactions repre-... 

There are fewer catalytic ribozymes compared to deoxyribozymes. Examples include a trara-splicing ribozyme, an alcohol dehydrogenase, a ligase capable of functioning at low temperature, " a ribozyme that will ligate the 5 -terminus of RNA to a polypeptide, a transcriptional activator and a tRNA aminoacylation catalyst. An RNA aptamer bearing 5 -CoA has been selected to catalyse thioester formation in the presence of biotin-AMP. In vitro selection has also been used to identify allosteric hairpin ribozymes, activated in the presence of short oligonucleotides, and a ribozyme that catalyses amide bond formation from a 2 -amino nucleotide. " ... 

NUCLEOSIDASES AND RELATED ENZYMES - LIGASES FORMING AMINOACYL-TRNA AND RELATED COMPOUNDS... 

The aminoacyl-tRNA ligases, of course, provide the mechanism by which an amino add is activated in the presence of ATP and an activating em me specie for that amino add. The reaction is a reversible one, and can be diagramm as follows [reaction (I)], bearing in mind that our former colleague Robert Loftfield may be correct in regarding the path from free amino acid to aminoacyl-tRNA in some instances to represent a concerted reaction mechanism, without a definitive intermediate. 

Translation involves two compartments the cytosol in which individual amino acids are enzymi-cally attached to their specific tRNAs by amino-acid-tRNA ligases (also called aminoacyl-tRNA synthetases) and the ribosomes in which the amino acids are correctly positioned according to the base sequence of a mRNA template and polymerized into polypeptide chains. 

In addition to their ability to load the tRNA with an amino acid, amino-acid-tRNA ligases are capable of recognizing inappropriate attachments, i.e. they have a proofreading function. If an incorrect amino acid has been attached, it may be removed by the hydrolysis of the enzyme-bound aminoacyl-adenylate intermediate. Once the anhydride linkage is cleaved, the amino acid and AMP dissociate from the active site so that the tRNA may be reloaded, hopefully with the correct molecule. The selectivity of these ligases is largely responsible for the maintenance of the fidelity of protein synthesis. 

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