TRNA synthetase compounds

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. 

Aminoacid-tRNA synthetases were isolated from spinach chloroplasts by methods developed at Jealott s Hill. Cytoplasmic aaTRS s from carrot were isolated by standard procedures. Compounds were assayed as inhibitors of the chloroplastic aaTRS s at a single rate, 10 pM. The reaction was followed by phosphate generation and the percentage inhibition determined using literature methods. The IC50 value for the spinach chloroplastic enzymes and inhibition data for carrot cytoplasmic aaTRS s were determined for compounds of interest. All compounds were tested for post-emergence herbicidal activity, at 0.125, or at 0.5, or at 2.0 k a in standard screens." ... 

The second mechanism to eliminate errors is at the level of amino-acid discrimination. The incorrect amino acid is removed either by hydrolyzing the aminoacyl adenylate compound after step 1 but before step 2, or as is the case with some aminoacyl-tRNA synthetases, the incorrect aminoacyl-tRNA is formed, but then hydrolyzed immediately after it is formed. 

Several other purine nucleotide derivatives with alkyl halide substituents have appeared in the literature, including adenosine S -chloromethane phosphonate, adenosine 5 -chloromethylpyrophosphate, and adenosine 5 -()8-bromoethane phosphonate) (287-291). These compounds have been evaluated as affinity labels of such nucleotide-binding enzymes as leucyl- and tryptophanyl-tRNA synthetases (leucine- and tryptophan-tRNA ligases), phosphorylase b, and cAMP-... 

Ribosomes were entrapped inside POPC vesicles together with poly(U), acting as template mRNA, t-RNA , Phe-tRNA-synthetase, Phe, and all low mw compounds required for the translation. Poly(Phe) is produced accordingly inside extruded vesicles. 

Some simpler analogues of the bacterial tyrosyl tRNA synthetase inhibitor SB-219383 have been prepared. Reaction of the nitrone 160, prepared from L-arabinose by intramolecular alkylation of an oxime, with the anion of Ph2C = NCH2C02Et gave the separable isomers of 161. These were converted to 162 and its diastereoisomer, and compounds with opposite chirality in the piperidine ring were made from D-arabinose. Compound 162 had much greater bioactivity than the other three isomers, also displaying selectivity for the... 

A three-component reaction of 2-diazoindan-l,3-dione (18), benzalde-hyde and substituted N-phenyhnaleimide gave in good yield a mixture of spirofuropyrroles 19 and 20 possessing three stereocenters in good yields (Scheme 6). These compounds 19 and 20 were reported to be potent tRNA synthetase inhibitors with IC50 of 0.6 and 0.004 M, respectively [68]. 

Esters of the substrate amino acids with alcohol residues of appreciable size are accepted by gramicidin S-synthetase and catalyze biosynthesis of gramicidin S (5). The activation mechanism for these compounds still needs to be elucidated. It is interesting that these findings are in contrast to the features observed for the ami noacyl adenylate activation of amino acids by tRNA synthetases in protein biosynthesis which are inhibited by substrate esters. From these results we infer that as in the tRNA synthetases the a-amino group of the substrate amino acids of gramicidin S synthetase is essential for their binding to the aminoacyl adenylate activation sites of the... 

Azetidine 2-carboxylic acid (39) is toxic both to plants and animals. When introduced into mung bean Phaseolus aureus), a plant tiiat does not normally have the compound, deleterious effects were noted. Azetidine 2-carboxylic acid is incorporated into protein instead of proline. Many of the Liliaceae have an altered prolyl tRNA synthetase that permits them to discriminate against 2-azetidine carboxylic acid (Rosenthal, 1991). 

One of these amino acids is azetidine-2-carboxylic acid (D 12.1), which in most organisms is accepted as substrate of prolyl-tRNA synthetase, an enzyme obligatory for protein biosynthesis. Azetidine-2-carboxylic acid is incorporated in proteins instead of L-proline, giving protein molecules with reduced biological efficiency. Prolyl-tRNA in plants which store azetidine-2-carbo-xylic acid, however, has a much higher substrate specificity and does not react with this compound. Even in the presence of azetidine-2-carboxylic acid therefore normal proteins will be formed. 

The bruchid beetle Caryedes brasiliensis, for instance, a specialized insect living on canavanine-containing seeds, possesses an arginyl-tRNA synthetase that, in contrast to the enzymes of most other organisms, discriminates against canavanine. Moreover the beetle is able to degrade cana-vanine to canaline and urea and to hydrolyze the latter compound to ammonia. 

For initiation an initiatory codon (AUG or GUG) is necessary in mRNA, as well as an initiatory tRNA capable of joining with methionine under the control of methionine-tRNA synthetase. The methionine residue is formilated at the NH2 group in the presence of transformilase. The initiation of protein synthesis requires, not only tRNA and rRNA, but also three protein compounds - Fj, F2, and F3, as well as GTP and the 30S subunit of ribosomes. 

One of the explanations for the toxic properties of some nonprotein amino acids is their ability to form anomalous proteins. When the structure of a particular compound is structurally analogous to its protein constituent, the nonprotein amino acid may be incorporated. The well-known case of canavanine substitution for arginine in insect proteins is due to the inability of most insects arginyl-tRNA synthetases to discriminate the two compounds (20). When canavanine containing food is consumed, proteins with disrupted tertiary structure are built and the insects may not survive. 

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