Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.1.1.20 (phenylalanyl-tRNA synthetase)
358 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The specificity of valyl-, phenylalanyl-, and tyrosyl-tRNA synthetases from yeast has been examined by a series of stringent tests designed to eliminate the possibility of artefactual interference. Valyl-tRNA synthetase, as well as activating a number of amino acid analogues, will accept alanine, cysteine, isoleucine, and serine in addition to threonine as substrates for both ATP-PPi exchange and transfer to some tRNAVal species. The transfer is not observed if atempts are made to isolate the appropriate aminoacyl-tRNAVal-C-C-A but its role in the overall aminoacylation can be suspected from both the formation of a stable aminoacyl-tRNAVal-C-C-A(3'NH2) compound and from the stoichiometry of ATP hydrolysis during the aminoacylation of the native tRNA. Similar tests with phenylalanyl-tRNA synthetase indicate that this enzyme will also activate and transfer other naturally occurring amino acids, namely, leucine, methionine, and tyrosine. The tyrosine enzyme, which lacks the hydrolytic capacity of the other two enzymes (von der Haar, F., & Cramer, F (1976) Biochemistry 15, 4131--4138) is probably absolutely specific for tyrosine. It is concluded that chemical proofreading, in terms of an enzymatic hydrolysis of a misacylated tRNA, plays an important part in maintaining the specificity in the overall reaction and that this activity may be more widespread than has so far been suspected.
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PMID:Aminoacyl-tRNA synthetases from yeast: generality of chemical proofreading in the prevention of misaminoacylation of tRNA. 35 80

Neither the tertiary structure nor the location of active sites are known for phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 structure), a member of class II aminoacyl-tRNA synthetases. In an attempt to detect the phenylalanine (Phe) binding site, two Escherichia coli PheRS mutant strains (pheS), which were resistant to p-fluorophenylalanine (p-F-Phe) were analysed genetically. The pheS mutations were found to cause Ala294 to Ser294 exchanges in the alpha subunits from both independent strains. This alteration (S294) resided in the well-conserved C-terminal part of the alpha subunit, precisely within motif 3, a typical class II tRNA synthetase sequence. We thus propose that motif 3 participates in the formation of the Phe binding site of PheRS. Mutation S294 was also the key for proposing a mechanism by which the substrate analogue p-F-Phe is excluded from the enzymatic reaction; this may be achieved by steric interactions between the para-position of the aromatic ring and the amino acid residue at position 294. The Phe binding site model was then tested by replacing the alanine at position 294 as well as the two flanking phenylalanines (positions 293 and 295) by a number of selected other amino acids. In vivo and in vitro results demonstrated that Phe293 and Phe295 are not directly involved in substrate binding, but replacements of those residues affected PheRS stability. However, exchanges at position 294 altered the binding of Phe, and certain mutants showed pronounced changes in specificity towards Phe analogues. Of particular interest was the Gly294 PheRS in which presumably an enlarged cavity for the para position of the aromatic ring allowed an increased aminoacylation of tRNA with p-F-Phe. Moreover, the larger para-chloro and para-bromo derivatives of Phe could interact with this enzyme in vitro and became highly toxic in vivo. The possible exploitation of the Gly294 mutant PheRS for the incorporation of non-proteinogenic amino acids into proteins is discussed.
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PMID:Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations. 194 71

Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[14C]tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe). The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the alpha subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].
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PMID:Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe. 282 80

Several tRNA's specific for a particular amino acid have been shown to exist in multiple, or isoaccepting, forms. There is considerable interest in establishing whether multiple aminoacyl-tRNA synthetases also exist. We present evidence that the cytoplasm of Neurospora crassa contains three chromatographically separable phenylalanyl-tRNA synthetases distinct from mitochondrial phenylalanyl-tRNA synthetase. In addition to differences in chromatographic properties the three enzymes exhibit different affinities, in Tris-Cl buffer, toward purified species of valine and alanine tRNA's isolated from Escherichia coli. The two major chromatographic fractions have very similar sedimentation characteristics, which makes a monomer-dimer relationship unlikely.
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PMID:Multiple phenylalanyl-transfer ribonucleic acid synthetase activities in the cytoplasm of Neurospora crassa. 525 12

Purified yeast phenylalanyl-tRNA synthetase can aminoacylate (yeast) tRNA(Phe), (wheat) tRNA(Phe), and (Escherichia coli) tRNA(1) (Val) (1, 2). We now report that this synthetase can also aminoacylate (E. coli) tRNA(Phe) and (E. coli) tRNA(1) (Ala). Highly purified (E. coli) tRNA(Phe) is heterologously aminoacylated to approximately 90% of the extent achieved with the homologous enzyme (crude E. coli phenylalanyl-tRNA synthetase). Pure (E. coli) tRNA(1) (Ala) (the major species) is heterologously aminoacylated to 70% of the extent achieved with the homologous synthetase (crude E. coli alanyl-tRNA synthetase).(E. coli) tRNA(Phe) is the fourth purified transfer RNA of known sequence to be shown to be an acceptable substrate for purified yeast phenylalanyl-tRNA synthetase. A comparison of these sequences shows that only one region is extremely similar in all four tRNAs. This region is located adjacent to the dihydrouridine loop, and consists of the nucleotides [Formula: see text] We conclude that this is the synthetase recognition site for yeast phenylalanyl-tRNA synthetase. This conclusion is further supported by partial fragment analysis of (E. coli) tRNA(1) (Ala).
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PMID:The yeast phenylalanyl-transfer RNA synthetase recognition site: the region adjacent to the dihydrouridine loop. 527 81

1. Phenylalanyl-tRNA synthetases have been partially purified from cotyledons of seeds of Aesculus californica, which contains 2-amino-4-methylhex-4-enoic acid, and from four other species of Aesculus that do not contain this amino acid. The A. californica preparation was free from other aminoacyl-tRNA synthetases, and the contaminating synthetase activity in preparations from A. hippocastanum was decreased to acceptable limits by conducting assays of pyrophosphate exchange activity in 0.5m-potassium chloride. 2. The phenylalanyl-tRNA synthetase from each species activated 2-amino-4-methylhex-4-enoic acid with K(m) 30-40 times that for phenylalanine. The maximum velocity for 2-amino-4-methylhex-4-enoic acid was only 30% of that for phenylalanine with the A. californica enzyme, but the maximum velocities for the two substrates were identical for the other four species. 3. 2-Amino-4-methylhex-4-enoic acid was not found in the protein of A. californica, so discrimination against this amino acid probably occurs in the step of transfer to tRNA, though subcellular localization, or subsequent steps of protein synthesis could be involved. 4. Crotylglycine, methallylglycine, ethallylglycine, 2-aminohex-4,5-dienoic acid, 2-amino-5-methylhex-4-enoic acid, 2-amino-4-methylhex-4-enoic acid, beta-(thien-2-yl)alanine, beta-(pyrazol-1-yl)alanine, phenylserine and m-fluorophenylalanine were substrates for pyrophosphate exchange catalysed by the phenylalanyl-tRNA synthetases of A. californica or A. hippocastanum. Allylglycine, phenylglycine and 2-amino-4-phenylbutyric acid were inactive.
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PMID:Properties and substrate specificities of the phenylalanyl-transfer-ribonucleic acid synthetases of Aesculus species. 549 4

Alanine and phenylalanine tRNA sequences were amplified by PCR from Arabidopsis thaliana nuclear DNA using degenerate oligonucleotides which introduced specific mutations into the acceptor stem. The aminoacylation of T7 RNA polymerase transcripts of these sequences was investigated in vitro using partially purified bean alanyl- or phenylalanyl-tRNA synthetase. In parallel, the in vivo activity of amber suppressor derivatives of these tRNAs was investigated in transient expression assays in tobacco protoplasts using a beta-glucuronidase (GUS) reporter gene containing a premature amber stop codon. The results show that mutation of the G3:U70 base pair to G3:C70 blocks aminoacylation of plant alanine tRNA, whilst conversion of the G3:C70 pair normally found in plant tRNA(Phe) to G3:U70 enables the mutated tRNA(Phe) to be a good substrate for alanyl-tRNA synthetase and impairs its aminoacylation with phenylalanine. In addition, the amber suppressor derivative of wild-type tRNA(Phe) showed very little suppressor activity in vivo, and was poorly aminoacylated with phenylalanine in vitro, suggesting that the anticodon is a major identity determinant for tRNA(Phe) in plant cells.
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PMID:Characterization of some major identity elements in plant alanine and phenylalanine transfer RNAs. 753 29

Alanine at position 294 (Ala294) within the motif 3 consensus of Escherichia coli phenylalanyl-tRNA synthetase alpha subunit has previously been implicated as a determinant of amino acid specificity. To characterize the role of Ala294, the catalytic effects of amino acid replacements at this position were tested with purified wild-type and mutant phenylalanyl-tRNA synthetases. We show that Ala294 is involved in amino acid binding and that it influences specificity as a determinant of binding pocket size. Replacement of Ala294 by either glycine or serine, thereby increasing or decreasing the size of the binding pocket, respectively, reduces affinity for phenylalanine. The Gly294 mutant shows a relaxed specificity toward synthetic para-halogenated phenylalanine analogues, the apparent dissociation constant Km increasing in direct relation to an increase of the van der Waals radius of the para group, thus confirming the role of position 294 in determining amino acid binding pocket size. For the substrate analogue p-chlorophenylalanine, attachment to tRNA and in vivo incorporation into cellular protein by the Gly294 mutant were demonstrated. Tyrosine activation was also improved with this mutant, but the resulting enzyme-Tyr-adenylate complex was rapidly hydrolyzed, indicating the presence of a proofreading mechanism in E. coli phenylalanyl-tRNA synthetase.
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PMID:Substrate specificity is determined by amino acid binding pocket size in Escherichia coli phenylalanyl-tRNA synthetase. 800 76

Streptococcus pneumoniae is a causative agent of nosocomial infections such as pneumonia, meningitis, and septicemia. Penicillin resistance in S. pneumoniae depends in part upon MurM, an aminoacyl-tRNA ligase that attaches L-serine or L-alanine to the stem peptide lysine of Lipid II in cell wall peptidoglycan. To investigate the exact substrates the translation machinery provides MurM, quality control by alanyl-tRNA synthetase (AlaRS) was investigated. AlaRS mischarged serine and glycine to tRNA(Ala), as observed in other bacteria, and also transferred alanine, serine, and glycine to tRNA(Phe). S. pneumoniae tRNA(Phe) has an unusual U4:C69 mismatch in its acceptor stem that prevents editing by phenylalanyl-tRNA synthetase (PheRS), leading to the accumulation of misaminoacylated tRNAs that could serve as substrates for translation or for MurM. Although the peptidoglycan layer of S. pneumoniae tolerates a combination of both branched and linear muropeptides, deletion of MurM results in a reversion to penicillin sensitivity in strains that were previously resistant. However, because MurM is not required for cell viability, the reason for its functional conservation across all strains of S. pneumoniae has remained elusive. We now show that MurM can directly function in translation quality control by acting as a broad specificity lipid-independent trans editing factor that deacylates tRNA. This activity of MurM does not require the presence of its second substrate, Lipid II, and can functionally substitute for the activity of widely conserved editing domain homologues of AlaRS, termed AlaXPs proteins, which are themselves absent from S. pneumoniae.
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PMID:Lipid II-independent trans editing of mischarged tRNAs by the penicillin resistance factor MurM. 2386 53

d-Amino acids are excluded at three different steps during protein synthesis: the aminoacylation of tRNA, binding of aminoacyl-tRNAs to EF-Tu, and selection of the aminoacyl-tRNA by the ribosome. We previously altered the enantioselectivity of tyrosyl-tRNA synthetase (TyrRS) by inserting the editing domain from phenylalanyl-tRNA synthetase (FRSed) between Gly 161 and Ile 162 in tyrosyl-tRNA synthetase (the editing domain hydrolyzes l-Tyr-tRNA but not d-Tyr-tRNA). In this paper, we test the hypothesis that the enantioselectivity of this TyrRS-FRSed chimera can be shifted further toward the formation of d-Tyr-tRNA by introducing activating mutations into the editing site. Yokoyama and colleagues previously identified six alanine substitutions in phenylalanyl-tRNA synthetase that increase its editing activity.1 We have introduced these alanine substitutions into TyrRS-FRSed in various combinations, generating 14 different variants. To analyze their editing activity, we developed a continuous, spectrophotometric, steady-state post-transfer editing assay in which l-Tyr-tRNA is generated in situ, resulting in the release of one molecule of AMP during each editing cycle. Post-transfer editing is monitored by coupling the release of AMP to the reduction of NAD(+) (via the actions of AMP deaminase and IMP dehydrogenase), resulting in an increase in absorbance at 340 nm. In general, TyrRS-FRSed variants containing two activating mutations are the most active, with additional alanine substitutions decreasing the activity of the editing domain. Linear free energy relationships indicate that high kcat values are correlated with high binding affinities for l-Tyr-tRNA. Lastly, competition assays indicate that at least one TyrRS-FRSed variant (F145A/S211A) preferentially aminoacylates tRNA with d-tyrosine, demonstrating that the enantioselectivity of tyrosyl-tRNA synthetase can be inverted using hyperactive editing domains.
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PMID:Hyperactive Editing Domain Variants Switch the Stereospecificity of Tyrosyl-tRNA Synthetase. 2706 38


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