Biochemical Characterization of a Mutant Isoleucyl-Transfer Ribonucleic Acid Synthetase from
            <i>Escherichia coli</i>
            K-12

Treiber, Gerda; Iaccarino, Maurizio

doi:10.1128/jb.107.3.828-832.1971

Cited by 20 publications

(5 citation statements)

References 11 publications

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“…Similar results have been reported by McLaughlin et al for yeast (17). These results are concordant with those obtained by using various analogues which inhibit the enzymatic activity of valyl-, isoleucylor leucyltransfer RNA synthetase (1,7,14,23,27,30). As predicted, some mutant strains which have amino acyl-transfer RNA synthetases resistant to those analogues have derepressed levels of the ilv enzymes (1, 7,27,30).…”

Section: Specific Activityasupporting

confidence: 86%

“…As predicted, some mutant strains which have amino acyl-transfer RNA synthetases resistant to those analogues have derepressed levels of the ilv enzymes (1, 7,27,30). Two analogues, O-methylthreonine and 4-thiaisoleucine, inhibit the isoleucyl-transfer RNA synthetase of E. coli (25,28,30). Both compounds inhibit the growth of Acinetobacter at low concentrations; however, the only ilv enzyme that is significantly derepressed in this organism is acetohydroxyacid synthetase.…”

Section: Specific Activityamentioning

confidence: 78%

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Enzymes of the Isoleucine-Valine Pathway in Acinetobacter

Twarog

1972

J Bacteriol

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Regulation of four of the enzymes required for isoleucine and valine biosynthesis in Acinetobacter was studied. A threeto fourfold derepression of acetohydroxyacid synthetase was routinely observed in two different wild-type strains when grown in minimal medium relative to cells grown in minimal medium supplemented with leucine, valine, and isoleucine. A similar degree of synthetase derepression was observed in appropriately grown isoleucine or leucine auxotrophs. No significant derepression of threonine deaminaseor transaminase B occurred in either wild-type or mutant cells grown under a variety of conditions. Three amino acid analogues were tested with wild-type cells; except for a two-to threefold derepression of dihydroxyacid dehydrase when high concentrations of aminobutyric acid were added to the medium, essentially the same results were obtained. Experiments showed that threonine deaminase is subject to feedback inhibition by isoleucine and that valine reverses this inhibition. Cooperative effects in threonine deaminase were demonstrated with crude extracts. The data indicate that the synthesis of isoleucine and valine in Acinetobacter is regulated by repression control of acetohydroxyacid synthetase and feedback inhibition of threonine deaminase and acetohydroxyacid synthetase.

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Section: Specific Activityasupporting

confidence: 86%

Section: Specific Activityamentioning

confidence: 78%

Enzymes of the Isoleucine-Valine Pathway in Acinetobacter

Twarog

1972

J Bacteriol

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“…Mutant proteins T242A and N250A were overexpressed and purified as previously described for the T242A mutant protein (21). Plasmids pTLH24 and pTLH25 were independently transformed and overexpressed in the E. coli strain MI1 (24,25). This strain contains a mutant ileS allele which introduces an F570S mutation into the synthetic active site of the chromosome-encoded IleRS (5).…”

Section: Methodsmentioning

confidence: 99%

Errors from Selective Disruption of the Editing Center in a tRNA Synthetase

2000

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Some aminoacyl-tRNA synthetases have two catalytic centers that together achieve fine-structure discrimination of closely similar amino acids. The role of tRNA is to stimulate translocation of a misactivated amino acid from the active site to the editing site where the misactivated substrate is eliminated by hydrolysis. Using isoleucyl-tRNA synthetase as an example, we placed mutations in the catalytic center for editing at residues strongly conserved from bacteria to humans. A particular single substitution and one double substitution resulted in production of mischarged tRNA, by interfering specifically with the chemical step of hydrolytic editing. The substitutions affected neither amino acid activation nor aminoacylation, with the cognate amino acid. Thus, because of the demonstrated functional independence of the two catalytic sites, errors of aminoacylation can be generated by selective mutations in the center for editing.

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“…These mutant enzymes were purified from strain Mil (Iaccarino & Berg, 1971;Treiber & Iaccarino, 1971), which contains a mutant ileS allele that allows for cell growth in isoleucine-rich media. Enzymes were plasmid overexpressed (10-20-fold in relation to chromosomally encoded enzyme) and purified.…”

Section: Methodsmentioning

confidence: 99%

Residues in a Class I tRNA Synthetase Which Determine Selectivity of Amino Acid Recognition in the Context of tRNA

Schmidt¹,

Schimmel²

1995

Biochemistry

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Certain aminoacyl-tRNA synthetases discriminate between closely similar amino acids by hydrolytic editing reactions in the presence of their cognate tRNA. An example is the class I isoleucyl-tRNA synthetase. We recently showed that a mutation which eliminates discrimination between isoleucine (Ile) and valine (Val) in the initial amino acid binding and activation steps had little effect on the hydrolytic editing of activated valine in the presence of isoleucine tRNA (tRNA(Ile)). The results showed that initial amino acid binding and discrimination are functionally independent of tRNA-dependent amino acid discrimination. In this work, we cross-linked (to isoleucyl-tRNA synthetase) a reactive analog of valine misacylated onto tRNA(Ile). Mutation of specific residues within a peptide segment identified by the cross-linking analysis severely affected discrimination of Val-tRNA(Ile) versus Ile-tRNA(Ile). The mutationally sensitive residues are part of an insertion into the catalytic domain and are themselves completely conserved among all known prokaryotic and eukaryotic sequences of the enzyme.

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Biochemical Characterization of a Mutant Isoleucyl-Transfer Ribonucleic Acid Synthetase from Escherichia coli K-12

Cited by 20 publications

References 11 publications

Enzymes of the Isoleucine-Valine Pathway in Acinetobacter

Enzymes of the Isoleucine-Valine Pathway in Acinetobacter

Errors from Selective Disruption of the Editing Center in a tRNA Synthetase

Residues in a Class I tRNA Synthetase Which Determine Selectivity of Amino Acid Recognition in the Context of tRNA

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