Isoleucyl tRNA

Bacterial ribosome function Aminoglycosides Tetracyclines Chloramphenicol Macrolides, azalides Fusidic acid Mupirocin Distort SOS ribosomal subunit Block SOS ribosomal subunit Inhibits peptidyl transferase Block translocation Inhibits elongation factor Inhibits isoleucyl-tRNA synthesis No action on 40S subunit Excluded by mammalian cells No action on mammalian equivalent No action on mammalian equivalent Excluded by mammalian cells No action on mammalian equivalent... 

The target of mupirocin is one of a group of enzymes which couple amino acids to their respective tRNAs for delivery to the ribosome and incorporation into protein. The particular enzyme inhibited by mupirocin is involved in producing isoleucyl-tRNA. The basis for the inhibition is a structural similarity between one end of the mupirocin molecule and isoleucine. Protein synthesis is halted when the ribosome encounters the isoleucine codon through depletion of the pool of isoleucyl-tRNA. 

Mupirocin is a topical antibiotic that inhibits isoleucyl tRNA synthetase with the subsequent inhibition of protein synthesis. Mupirocin has become a mainstay in the treatment of Staph, aureus infection and colonization during hospital outbreaks, and it is in this organism that acquired resistance has arisen (Gilbart etal. 1993). 

Gilbart J., Perry CR. Slocombe B. (1993) High-level mupirocin resistance in Staphylococcus aureus evidence for two distinct isoleucyl-tRNA synthetases. Antimicrob Agents Chemother, 31, 32-38. Godfrey A.J. Bryan L.E. (1984) Intrinsic resistance and whole cell factors contributing to antibiotic resistance, hv. Antimicrobial Drug Resistance (ed. L.E. Bryan), pp. 113-145. New York Academic Press. 

T. Kohno, D. Kohda, M. Haruki, S. Yokoyama, and T. Miyazawa, Non-protein amino acid furanomycin, unlike isoleucine in chemical structure, is changed to isoleucine tRNA by isoleucyl-tRNA synthetase and incorporated into protein. J. Biol. 

A recent example of the success of the bisubstrate, transition state design approach comes from the work of Pope and coworkers (Pope et al., 1998a-c Brown et al., 2000) on the design of inhibitors of bacterial isoleucyl tRNA synthetase... 

Hughes, J., and Mellows, G. (1980). Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase. Biochem. J. 191, 209—219. 

Mupirocin, pseudomonic acid, is a product of Pseudomonas fluorescens discovered by Fuller etal. It is an inhibitor of isoleucyl-transfer ribonucleic acid (isoleucyl-tRNA) synthetase. 

This enzyme [EC 6.1.1.5], also referred to as isoleu-cinedRNA ligase, catalyzes the reaction of isoleucine with tRNA and ATP to produce isoleucyl-tRNA , AMP, and pyrophosphate. 

ISOLEUCYL-tRNA SYNTHETASE, AMINO-ACYL tRNA SYNTHETASES Isolobal,... 

Naturally derived from fermentation using Pseudomonas fluorescens and active against MRSA and some other grampositive bacteria. Acts by blocking the activity of isoleucyl-tRNA synthetase in bacteria. This enzyme is necessary for bacteria to synthesize proteins. 

This editing mechanism for isoleucyl-tRNA synthetase was demonstrated directly in 1998 by X-ray crystallography on complexes of the enzyme with L-isoleucine and L-valine. Both substrates fit into the ATP-requir-ing synthetic site but neither isoleucine nor isoleucyl-tRNA will fit into the editing site which is located in an adjacent (3-barrel domain.104 105 Proofreading steps based on differing chemical properties as well as size can also be visualized.103 106... 

Many antibiotics, which inhibit protein synthesis, do not bind to ribosomes but block any of a variety of vital chemical processes needed for growth. Among them are pseudomonic acid, which inhibits isoleucyl-tRNA synthetase from many gram-positive bacteria.1111/VV Rapamycin, best known as an immunosuppressant (Box 9-F), inhibits phosphoinositide-3-kinase and also phosphorylation of the cap-binding protein 4G, a component of the eukaryotic initiation factor complex (Fig. 29-11 ).ww The bacterial enzyme peptide deformylase, which is absent from the human body, has been suggested as a target for design of synthetic antibiotics. 01... 

Discrimination between some pairs of tRNAs depends entirely on the anticodon sequence. For example, tRNAMet contains the anticodon CAU. That for a minor tRNAIle is the same except that the cytosine has been posttranscriptionally modified by covalent linkage of a molecule of lysine via its e-amino group to C2 of the cytosine. The latter base (Iysidine) is correctly recognized by E. coli isoleucyl-tRNA synthetase but, if the cytosine is unmodified, it is aminoacylated by methionyl-tRNA synthetase.192 In most instances the acceptor specificity, or tRNA identity, is not determined solely by the anticodon sequence. Thus, when a methionine initiator tRNA was modified to contain a tryptophan anticodon, it was only partially charged with tryptophan in vivo. However, when A73 of the methionine tRNA was also converted to G73, only tryptophan was inserted.193 Nucleotide 73 (Fig. 

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.)...

The structure of the isoleucyl-tRNA synthetase (IleRS) from Thermus ther-mophilus (1045 residues, Mr 120 000) has been solved, as well as its complexes with lie and Val.17 The protein contains a nucleotide binding fold (Chapter 1) that binds ATR The fold has two characteristic ATP binding motifs His-54-Val-55-Gly-56-His-57 and Lys-591-Met-592-Ser-593-Lys-594. In the L-Ile-IleRS complex, a single He is bound at the bottom of the ATP cleft, with the hydrophobic side chain in a hydrophobic pocket, surrounded by Pro-46, Trp-518, and Trp-558. L-Leucine cannot fit into this pocket because of the steric hindrance of one of its terminal methyl groups. Larger amino acids are similarly excluded from this site. In the l-Val-IleRS complex, Val is bound to the same site, but the... 

Uracil is removed from the DNA by a uracil glycosidase which excises the base from the sugar ring. This activity is analogous to the hydrolytic activity of the isoleucyl-tRNA synthetase toward Val-tRNAIle. In both cases the hydrolytic site is too small by the size of one methylene group to accommodate the substrate that is to be left intact. In DNA synthesis, the editing is performed by a separate enzyme, since the editing can wait until after polymerization. As this luxury is not permitted in protein synthesis, the hydrolytic function is on the synthetase, so that correction can occur before the misacylated tRNA leaves the enzyme. 

In this case, / = / /". For the isoleucyl-tRNA synthetase, / 150, so any subsequent increase in specificity is limited to a factor of less than 15 if the cost is to be kept tolerable. In fact, the measured cost is less than 0.05, and it may be predicted from the cost-selectivity equation that if the Hopfield mechanism were operating it would be increasing specificity by a factor of less than 8.5.55 We show next that double sieving is not limited in the same way. 

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) ... 

Fiqure 13.1 The binding cavity at the active site of the isoleucyl-tRNA synthetase must be able to bind valine as it binds the larger isoleucine. The active site of the valyl-tRNA synthetase cannot exclude threonine, because it is isosteric with valine. 

Pope, A.J. et al. 1998. Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus II mechanism of inhibition by reaction intermediate and pseudomonic acid analogues studied using transient and steady-state kinetics. J. Biol. Chem. 273, 31691-31701. 

Prompted by the herbicidal activity of monate derivatives, which are inhibitors of prokaryotic isoleucyl-tRNA synthetase (ITRS), several analogues of structure 1 were prepared and their biological activities characterised to establish if there was a common SAR between herbicidal activity and aaTRS inhibitor activity. 

Table 1 Percentage inhibition of a range of spinach chloroplast aaTRS s by sulfamoyladenosines at lOpM (ND = Not Determined, LTRS = leucyl-tRNA synthetase, ITRS = isoleucyl-tRNA synthetase, VTRS = valyl-tRNA synthetase, ATRS = Alanyl-tRNA synthetase, GTRS = glycyl-tRNA synthetase)...

Synthetase mechanism. The formation of isoleucyl-tRNA proceeds through the reversible formation of an enzyme-bound He-AMP intermediate. Predict whether 32p-labeled ATP is formed from 32ppj when each of the following sets of components is incubated with the specific activating enzyme ... 

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