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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)

Phenylalanyl-tRNA synthetase from baker's yeast in the presence of phenylalanine or other amino acids misactivated by the enzyme, ATP, and low concentrations of Zn2+ is able to hydrolyze ATP to AMP and PPi very efficiently. After dialysis of the enzyme against ethylenediaminetetraacetic acid (EDTA), this amino acid dependent but tRNAPhe-independent hydrolysis is suppressed to negligible levels. The ATP hydrolysis can be restored by the addition of Zn2+ to the EDTA-dialyzed enzyme. During aminoacylation of tRNAPhe the Zn2+-induced ATP hydrolysis parallels the aminoacylation reaction, leading to nonstoichiometric production of AMP. Mechanistically, we conclude that Zn2+ can be bound to phenylalanyl-tRNA synthetase and can influence the stability of ATP if an activatable amino acid is present. The influence of Zn2+, if any, on the aminoacylation of tRNAPhe is not known. In practice, this side reaction is of the utmost importance in all cases in which the fate of ATP during aminoacylation is followed, especially if the stoichiometry of ATP consumption in relation to Phe-tRNAPhe formation has to be determined.
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PMID:A novel enzymatic activity of phenylalanyl transfer ribonucleic acid synthetase from baker's yeast: zinc ion induced transfer ribonucleic acid independent hydrolysis of adenosine triphosphate. 676 76

Adenosine or CpCpA trinucleoside diphosphate can be aminoacylated by phenylalanyl-tRNA synthetase [L-phenylalanine:tRNAPhe ligase (AMP forming), EC 6.1.1.20] when the reaction takes place in the presence of tRNAPhe deprived of its 3' adenosine or pCpCpA terminus. This shows that, upon interaction with tRNA, a structural alteration of the enzyme's active site is achieved. This process may be a determining step in the specificity of the aminoacylation reaction.
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PMID:Conformational activation of the yeast phenylalanyl-tRNA synthetase catalytic site induced by tRNAPhe interaction: triggering of adenosine or CpCpA trinucleoside diphosphate aminoacylation upon binding of tRNAPhe lacking these residues. 701 39

"Induced hydrolysis" a new hydrolytic activity, was found by measuring AMP-production during aminoacylation of tRNAPhe-CCA by yeast phenylalanyl-tRNA synthetase in the presence of tRNAPhe-CC under conditions of low ionic strength at pH 8.5. Experiments using the elongation factor Tu . GTP provide evidence that transfer of phenylalanine to the tRNAPhe-CCA is followed by rapid hydrolysis in the presence of tRNAPhe-CC. A simple mechanism shows good agreement with the experimental data.
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PMID:Induced hydrolytic activity of yeast phenylalanyl-tRNA synthetase by tRNAPhe-CC. 704 11

The transfer of amino acid to tRNA by Escherichia coli phenylalanyl-tRNA synthetase (PheRS) was studied using replacements of Ala294 in the alpha subunit previously shown to have modified amino acid specificity. Steady-state analysis of tRNA charging showed little difference between wild-type and mutants, whereas pre-steady-state analysis revealed higher rates of tRNA charging by both the A294S PheRS-phenylalanyl adenylate and the A294G PheRS-p-Cl-phenylalanyl adenylate. The decrease in energy required for the formation of the transition state of amino acid transfer in these mutants could be related to a weaker binding of the amino acid in the aminoacyl adenylate complex. Thus a compromise appears to exist between amino acid activation and tRNA charging, because slowing down the first step increases the rate of the second step, possibly as a result of decreased stability of the PheRS.amino acid-AMP complex.
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PMID:Increased rates of tRNA charging through modification of the enzyme-aminoacyl-adenylate complex of phenylalanyl-tRNA synthetase. 784 18

The crystal structures of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) complexed with phenylalanine and phenylalaninyl-adenylate (PheOH-AMP), the synthetic analogue of phenylalanyl-adenylate, have been determined at 2.7A and 2.5A resolution, respectively. Both Phe and PheOH-AMP are engulfed in the active site cleft of the catalytic alpha-subunit of PheRS, and neither makes contact with the PheRS beta-subunit. The conformations and binding of Phe are almost identical in both complexes. The recognition of Phe by PheRS is achieved through a mixture of multiple van der Waals interactions and hydrogen bonds. The side-chain of the Phe substrate is sandwiched between the hydrophobic side-chains of Phealpha258 and Phealpha260 on one side, and the main-chain atoms of the two adjacent beta-strands on the other. The side-chains of Valalpha261 and Alaalpha314 form the back wall of the amino acid binding pocket. In addition, PheRS residues (Trpalpha149, Seralpha180, Hisalpha178, Argalpha204, Glnalpha218, and Glualpha220) form a total of seven hydrogen bonds with the main-chain atoms of Phe. The conformation of PheOH-AMP and the network of interactions of its AMP moiety with PheRS are reminiscent of the other class II synthetases. The structural similarity between PheRS and histidyl-tRNA synthetase extends to the amino acid binding site, which is normally unique for each enzyme. The complex structures suggest that the PheRS beta-subunit may affect the first step of the reaction (formation of phenylalanyl-adenylate) through the metal-mediated conserved alpha/beta-subunit interface. The modeling of tyrosine in the active site of PheRS revealed no apparent close contacts between tyrosine and the PheRS residues. This result implies that the proofreading mechanism against activated tyrosine, rather than direct recognition, may play the major role in the PheRS specificity.
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PMID:Crystal structures of phenylalanyl-tRNA synthetase complexed with phenylalanine and a phenylalanyl-adenylate analogue. 1009 59

The crystal structure of phenylalanyl-tRNA synthetase (PheRS) from Thermus thermophilus, a class II aminoacyl-tRNA synthetase, complexed with phenylalanyl-adenylate (Phe-AMP) was determined at 2.6 A resolution. Crystals of native PheRS were soaked in a solution containing phenylalanine and ATP in the presence of Mn(2+) ions. The first step of the aminoacylation reaction proceeds within the crystals, resulting in Phe-AMP formation at the active site. Specific recognition of the phenylalanine portion of the Phe-AMP is achieved by interactions of the phenyl ring of Phe-AMP with two neighbouring residues, Phealpha258 and Phealpha260. No manganese ions were observed within the active site; their role in the formation of the transition state may be assigned to a number of polar residues and water molecules. In the anomalous Fourier difference map, a divalent metal ion was detected at the interface of the alpha- and beta-subunits at a short distance from motif 3 residues participating in the substrate binding. A sulfate ion, which was identified on the protein surface, may mediate the interactions of PheRS with DNA. Visible conformational changes were detected in the active-site area adjacent to the position of the Phe-AMP, compared with the structure of PheRS complexed with a synthetic adenylate analogue (phenylalaninyl-adenylate). Based on the known structures of the substrate-free enzyme and its complexes with various ligands, a general scheme for the phenylalanylation mechanism is proposed.
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PMID:Structure at 2.6 A resolution of phenylalanyl-tRNA synthetase complexed with phenylalanyl-adenylate in the presence of manganese. 1167 17

The crystal structure of the ternary complex of (alphabeta)(2) heterotetrameric phenylalanyl-tRNA synthetase (PheRS) from Thermus thermophilus with cognate tRNA(Phe) and a nonhydrolyzable phenylalanyl-adenylate analogue (PheOH-AMP) has been determined at 3.1 A resolution. It reveals conformational changes in tRNA(Phe) induced by the PheOH-AMP binding. The single-stranded 3' end exhibits a hairpin conformation in contrast to the partial unwinding observed previously in the binary PheRS.tRNA(Phe) complex. The CCA end orientation is stabilized by extensive base-specific interactions of A76 and C75 with the protein and by intra-RNA interactions of A73 with adjacent nucleotides. The 4-amino group of the "bulged out" C75 is trapped by two negatively charged residues of the beta subunit (Glubeta31 and Aspbeta33), highly conserved in eubacterial PheRSs. The position of the A76 base is stabilized by interactions with Hisalpha212 of motif 2 (universally conserved in PheRSs) and class II-invariant Argalpha321 of motif 3. Important conformational changes induced by the binding of tRNA(Phe) and PheOH-AMP are observed in the catalytic domain: the motif 2 loop and a "helical" loop (residues 139-152 of the alpha subunit) undergo coordinated displacement; Metalpha148 of the helical loop adopts a conformation preventing the 2'-OH group of A76 from approaching the alpha-carbonyl carbon of PheOH-AMP. The unfavorable position of the terminal ribose stems from the absence of the alpha-carbonyl oxygen in the analogue. Our data suggest that the idiosyncratic feature of PheRS, which aminoacylates the 2'-OH group of the terminal ribose, is dictated by the system-specific topology of the CCA end-binding site.
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PMID:The crystal structure of the ternary complex of phenylalanyl-tRNA synthetase with tRNAPhe and a phenylalanyl-adenylate analogue reveals a conformational switch of the CCA end. 1693 9

All class II aminoacyl-tRNA synthetases (aaRSs) are known to be active as functional homodimers, homotetramers, or heterotetramers. However, multimeric organization is not a prerequisite for phenylalanylation activity, as monomeric mitochondrial phenylalanyl-tRNA synthetase (PheRS) is also active. We herein report the structure, at 2.2 A resolution, of a human monomeric mitPheRS complexed with Phe-AMP. The smallest known aaRS, which is, in fact, 1/5 of a cytoplasmic analog, is a chimera of the catalytic module of the alpha and anticodon binding domain (ABD) of the bacterial beta subunit of (alphabeta)2 PheRS. We demonstrate that the ABD located at the C terminus of mitPheRS overlaps with the acceptor stem of phenylalanine transfer RNA (tRNAPhe) if the substrate is positioned in a manner similar to that seen in the binary Thermus thermophilus complex. Thus, formation of the PheRS-tRNAPhe complex in human mitochondria must be accompanied by considerable rearrangement (hinge-type rotation through approximately 160 degrees) of the ABD upon tRNA binding.
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PMID:The tRNA-induced conformational activation of human mitochondrial phenylalanyl-tRNA synthetase. 1861 82

At least one bisaminoacyl-tRNA is synthesized in nature (by Thermus thermophilus phenylalanyl-tRNA synthetase), and many disubstituted tRNAs have been prepared in vitro. Such misacylated tRNAs are able to participate in protein synthesis, even though they lack the free 2'-OH group of the 3'-terminal adenosine moiety. Their ready participation in protein synthesis implies significant chemical reactivity. The basis for this reactivity has been documented previously. Surprisingly, the aminoacyl moieties of these tRNAs also exhibit exceptional chemical stability. In the present report, bisaminoacylated nucleotides are investigated computationally and experimentally to define the basis for the stability of such species. Molecular modeling of bisalanyl-AMP in the absence of solvent and in the presence of a limited number of water molecules revealed two common features among the low-energy structures. The first was the presence of H-bonding interactions between the two aminoacyl moieties. The second was the presence of a H-bonding interaction between the 2'-O-alanyl moiety and the N-3 atom of the adenine nucleobase, typically mediated through a water molecule. The prediction of an interaction between an aminoacyl moiety and the adenine nucleobase was confirmed experimentally by comparing the behavior of bisalanyl-AMP and bisalanyl-UMP in the presence of model nucleophiles. This study suggests a possible role for the adenosine moiety at the 3'-end of aminoanyl-tRNAs in controlling the stability and reactivity of the aminoacyl moiety and has important implications for the reactivity and stability of normal aminoacyl-tRNAs.
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PMID:Structural basis for the exceptional stability of bisaminoacylated nucleotides and transfer RNAs. 2164 86

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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