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At least 19 recordsLinked to original sources
PubMed1976

Phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Substrate specificity with regard to ATP analogs and mechanism of the aminoacylation reaction.

Eighteen analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Four compounds are substrates for phenylalanyl-tRNA synthetase, five for seryl-tRNA synthetase, one compound is an inhibitor for both enzymes; their Km and Ki and V values have been determined. The substrate specificity shows that for the catalytic action of both enzymes with these substrates positions 6, 7, 8 and 9 of the purine moiety and positions 2' and 3' of the ribose moiety are important.

Adenosine Triphosphate↗
PubMed1976

Hydrolytic action of aminoacyl-tRNA synthetases from baker's yeast: "chemical proofreading" preventing acylation of tRNA(I1e) with misactivated valine.

Phe-tRNAPhe-C-C-A, Val-tRNAVal-C-C-A, and Ile-tRNAIle-C-C-A, which accept their amino acid on the 2'-OH of the 3'-terminal adenosine, are hydrolyzed readily by their aminoacyl-tRNA synthetase. If the 3'terminal adenosine in these tRNAs is replaced by either 3'-deoxyadenosine or formycin, little if any hydrolysis can be observed. Correspondingly Ser-tRNASer-C-C-A which accepts serine on the 3'-OH of the 3'-terminal adenosine is hydrolyzed by seryl-tRNA synthetase, whereas Ser-tRNASer-C-C-2'dA and Ser-tRNASer-C-C-F are not. Tyr-tRNATyr-C-C-A and all modified Tyr-tRNATyr-C-C-N, which can accept tyrosine on either the 2'OH or the 3'-OH of the 3'terminal adenosine, are not hydrolyzed by tyrosyl-tRNA synthetase. The data can be rationalized assuming that hydrolysis takes place only if the amino acid is bound to the nonaccepting OH and hence is not positioned at the amino acid binding site upon formation of the complex between aminoacyl-tRNA and aminoacyl-tRNA synthetase. In the formycin-carrying tRNA, the amino acid bound to the nonaccepting OH seems to be inaccessible to the enzymatic groups responsible for hydrolysis. Val-tRNAIle-C-C-3'dA and Ile-tRNAIle-C-C-3'DA cannot be hydrolyzed by isoleucyl-tRNA synthetase. Val-tRNAIle-C-C-A is hydrolyzed by the enzyme five times more rapidly than Ile-tRNAIle-C-C-A. Whereas Ile-tRNAIle-C-C-F is absolutely stable, Val-tRNAIle-C-C-F si hydrolyzed immediately. As shown by the earlier finding that valine misactivated by isoleucyl-tRNA synthetase cannot be permanently transferred to tRNAIle-C-C-A but to tRNAIle-C-C-3'dA, the 3'-OH is essential for preventing transfer of misactivated valine. It thus appears that valine is hydrolyzed off Val-tRNAIle-C-C-N if it is bound to the accepting 2'-OH in the binding site for isoleucine. A hypothesis is offered attempting to explain the experimental observations in mechanistic terms. We consider the hydrolytic action of the aminoacyl-tRNA synthetases as a general mechanism of "chemical proofreading" in the protein biosynthesis.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Aminoacyl adenylate, a normal intermediate or a dead end in aminoacylation of transfer ribonucleic acid.

The shape of the time curve for the aminoacylation of tRNA has been investigated using five different amino acid:tRNA ligases. Four of these enzymes showed a lag in the time curve during the early phase of the first catalytic turnover of the enzyme. In each case, the lag period could be abolished by preincubating the ligase with amino acid, ATP, and Mg2+ under conditions known to give an aminoacyl adenylate-enzyme complex. With all five ligases the steady state rate of transfer from the preformed aminoacyl-adenylate complex to tRNA was approximately the same as that of the overall reaction.

Adenosine Monophosphate↗
PubMed1979

Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. IV. Mutants with increased levels of leucyl- or seryl-tRNA synthetase.

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile leucyl-tRNA synthetase and seryl-tRNA synthetase were selected for growth at 40 degrees C. Among these, strains were found with increased levels of both thermolabile synthetases. Two distinct genetic loci were found responsible for enzyme overproduction. leuR, located near xyl, causes elevated levels of leucyl-tRNA synthetase; while serR, located near leu, causes elevated levels of seryl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1976

[Isolation and characterization of seryl- and phenylalanyl-tRNA synthetase from yeast (author's transl)].

A procedure for the simultaneous isolation of seryl- and phenylalanyl-tRNA synthetase from yeast is described. In addition some other synthetases as well as tRNA nucleotidyltransferase can be obtained in an enriched state. The isolated seryl- and phenylalanyl-tRNA synthetases were compared to earlier preparations with respect to purity, specific activity, and structure. Previous investigations with fluorescence spectroscopy and kinetic methods were complemented and extended by experiments on the specificity of aminoacylation and on the isolation, by sucrose gradient centrifugation, of complexes between synthetase and tRNA or tRNA fragments. A protection of synthetases against inactivation by addition of substrates was observed. The dissociation of seryl-tRNA synthetase, at low concentrations, into monomer subunits was investigated by chemical modification with bifunctional reagents and by kinetic experiments. By modification of SH-groups fluorescent dyes were incorporated into both, seryl- and phenylalanyl-tRNA synthetase which retained most of their activity. The binding of tRNAPhe to phenylalanyl-tRNA synthetase which had been modified with pyrene maleimid was followed by fluorescence intensity measurements.

Alanine-tRNA Ligase↗
PubMed1976

Growth rate modulation of four aminoacyl-transfer ribonucleic acid synthetases in enteric bacteria.

The specific activities of arginyl- glutamyl- seryl-, and valyl-transfer ribonucleic acid (tRNA) synthetases were measured in the wild-type and mutant strains of Salmonella typhimurium LT2 and Escherichia coli B/r. In media restricted only by carbon and energy source availability, the specific activities of all four enzymes were proportional to the growth rate, with the exception of seryl-tRNA synthetase in S. typhimurium, which remained essentially constant. Structural gene densities were calculated for these four enzymes and were found not to account for the variation of specific activity with growth rate.

Amino Acids↗
PubMed1978

Yellow lupin (Lupinus luteus) aminoacyl-tRNA synthetases. Isolation and some properties of enzyme-bound valyl adenylate and seryl adenylate.

As a continuation of our studies on plant (yellow lupin, Lupinus luteus) aminoacyl-tRNA synthetases we describe here formation and some properties of valyl-tRNA synthetase-bound valyl adenylate (EVal(Val-AMP)) and seryl-tRNA synthetase-bound seryl adenylate (ESer(Ser-AMP)). Valyl-tRNA synthetase-bound valyl adenylate was detected and isolated by several approaches in the pH range 6--10. In that range inorganic pyrophosphatase increases the amount of valyl adenylate by factor 1.8 regardless of pH. 50% of valine from the EVal(Val-AMP) complex isolated by Sephadex G-100 gel filtration was transferred to tRNA with a rate constant greater than 4 min-1 (pH 6.2, 10 degrees C). The ratio of valine to AMP in the enzyme-bound valyl adenylate is 1 : 1 and it is not changed by the presence of periodate-oxidized tRNA. In contrast to enzyme-bound valyl adenylate, formation of ESer(Ser-AMP) is very sensitive to pH. Inorganic pyrophosphatase increases the amount of seryl adenylate by a factor 6 at pH 8.0 and 30 at pH 6.9 60% of serine from the ESer(Ser-AMP) complex was transferred to tRNA with a rate constant greater than 4 min-1 (pH 8.0, 0 degrees C). The ratio of serine to AMP in the enzyme-bound seryl adenylate is 1 : 1. The rate of synthesis of the enzyme-bound aminoacyl adenylates was measured by ATP-PPi exchange. Michaelis constants for the substrates of valyl-tRNA and seryl-tRNA synthetases in ATP-PPi exchange were determined. Effects of pH, MgCl2 and KCl on the initial velocity of aminoacyl adenylate formation are described. For comparison, catalytic indices in the aminoacylation reactions catalyzed by both lupin enzymes are given and effects of pH, MgCl2 and KCl on tRNA aminoacylation are presented as well. Under some conditions, e.g. at low pH or high salt concentration, lupin valyl-tRNA and seryl-tRNA synthetase are active exclusively in ATP-PPi exchange reaction.

Adenosine Monophosphate↗
PubMed1978

Yeast seryl tRNA synthetase: two sets of substrate sites involved in aminoacylation.

Seryl tRNA synthetase from Saccharomyces Carlsbergensis C836 contains two sets of sites for tRNASer, L-serine, and Mg2+-ATP, both of which are involved in aminoacylation. This is based on the following experimental results: (a) at low serine concentrations, second order kinetics in tRNASer are observed; (b) biphasic kinetics result when the amino acid is the varied substrate indicating anticooperative binding of two serine molecules to the synthetase; (c) when two molecules of serine are bound the rate of aminoacylation increases strongly and becomes first order in tRNASer; (d) the involvement of more than one site for Mg2+ and ATP is deduced from systematic variations of the concentrations of Mg2+ and ATP. Implications of the anticooperative binding of the substrates for possible reaction mechanisms are discussed. The results indicate that under normal conditions, the activity of seryl tRNA synthetase is regulated mainly by tRNASer while at high serine concentrations regulation by the amino acid itself prevails.

Adenosine Triphosphate↗
PubMed1978

Yeast seryl tRNA synthetase: interactions between the ATP binding site and the sites for tRNASer and L-serine.

T1 ribonuclease digestion of yeast tRNASer in the presence of seryl tRNA synthetase was used for monitoring the relationship between the substrate binding sites on the synthetase. It was found that (a) ATP displaces the tRNA from the synthetase with an effector affinity constant corresponding to the Km for ATP of 10 micron; (b) AMP and a number of nucleoside triphosphates, while influencing the rate of aminoacylation, do not displace the tRNA from the enzyme; (c) ADP and PPi inhibit the aminoacylation and the binding of tRNASer; (d) adenylyl diphosphonate is bound to the synthetase and lowers the protection of the tRNA against the nuclease attack in a similar way as does ATP; (e) interactions between the sites of L-serine and tRNASer could only be shown when both sites for serine were saturated and, in addition, the ATP analog or ADP was present. It is concluded that in seryl tRNA synthetase binding sites for ATP interact with the ones for tRNA as well as with the ones for serine. These findings contribute to the understanding of the mechanism of aminoacylation.

Adenosine Triphosphate↗
PubMed1975

The plant aminoacyl-tRNA synthetases. Purification and characterization of valyl-tRNA, tryptophanyl-tRNA and seryl-tRNA synthetases from yellow-lupin seeds.

Valyl-tRNA, tryptophanyl-tRNA, and seryl-tRNA synthetases from yellow lupin seeds Lupinus luteus were purified to homogeneity by ammonium sulfate fractionation, hydrophobic chromatography on aminohexyl-Sepharose column and affinity chromatography on tRNA-Sepharose column. Valyl-tRNA synthetase consists of one polypeptide chain of molecular weight 125000 as judged by Sephadex G-200 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agent. Seryl-tRNA synthetase, Mr equals 110000, is composed of two 55000-Mr subunits. Tryptophanyl-tRNA synthetase exhibits molecular weight of 200000 on Sephadex G-200 and 37000 in dodecylsulfate-polyacrylamide gel electrophoresis. This indicates that tryptophanyl-tRNA synthetase consists of several subunits (probably four). Since the seryl-tRNA synthetase exhibits the same mobility on dodecylsulfate-polyacrylamide gels both in the presence and absence of reducing agent it is concluded that there is no covalent bond(s) between the subunits of the enzyme. There is also no covalent bond(s) between the subunits of tryptophanyl-tRNA synthetase. Effect of anti-sulfhydryl reagents, monovalent salts, pH and different buffers on activity of the three synthetases is described. Kinetic constants for the substrates of the synthetases are also given. dATP is a substrate for seryl-tRNA synthetase but not for valyl-tRNA and tryptophanyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Nuclease digestion of synthetase x tRNA complexes.

Phenylalanyl-tRNA and seryl-tRNA synthetase protect strongly though not completely their cognate tRNAs against nuclease attack, as had been shown previously. In an investigation of the mechanism of protection it was demonstrated that the low susceptibility of phenylalanyl-tRNA-synthetase x tRNA-Phe complexes to nucleases is due to free tRNA present in equilibrium with synthetase. The equilibrium can be shifted by an excess of synthetase or by dilution of the complex. It therefore appears that synthetase competes with the nuclease for free tRNA. Degradation of the complex is low, however, because under the conditions of partial digestion the synthetase has a greater affinity for the tRNA than does the nuclease. Fragmented tRNAs, as they are formed during partial nuclease digestion, bind to synthetase to different degrees. tRNA-Phe with a lesion in the dihydrouridine loop binds very poorly whereas a nick in the anticodon loop reduces the strength of binding to a much lesser extent. In a systematic study of the stoichiometry of protection it was confirmed that under standard conditions one phenylalanyl-tRNA synthetase protects one tRNA-Phe and one seryl-tRNA synthetase two tRNA-Ser molecules against nuclease attack. Under certain conditions, however, (concentration of the complex higher than 10 mu-M, or alternately in buffers of low ionic strength) it is observed that phenylalanyl-tRNA synthetase binds up to 1.6 molecules tRNA-Phe. In the serine system, these special conditions do not affect the binding properties of seryl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1976

On the interaction of seryl-tRNA synthetase with tRNA Ser. A contribution to the problem of synthetase-tRNA recognition.

By following the tryptophan fluorescence of yeast seryl-tRNA synthetase on addition of tRNA Ser it was observed that the number of binding sites for tRNA decreases from two to one with increasing temperature, ATP or KCl concentration. Concomitantly a considerable decrease of the apparent binding constant was observed. The variation in the number of binding sites is explained by the presence of at least one temperature and ionic strength sensitive binding site and one temperature and ionic strength independent binding site. Relaxation kinetic experiments revealed two binding processes: a fast one depending on tRNA concentration and ionic strength and a slow one, which appeared to be independent of tRNA concentration and ionic strength. Enzyme kinetic studies showed that the activity of seryl-tRNA synthetase strongly depends on the KCl concentration and exhibits a maximum at 0.2 M KCl. Based on the data from relaxation and enzyme kinetic experiments a model is suggested for the recognition process involving a first unspecific step where all tRNAs, cognate and non-cognate, are bound to the synthetase (scanning step). The identification of the cognate tRNA is then performed at the recognition site by a conformational transition of the tRNA . synthetase complex (identification step).

Amino Acyl-tRNA Synthetases↗
PubMed1976

Distinct steps in the specific binding of tRNA to aminoacyl-tRNA synthetase. Temperature-jump studies on the serine-specific system from yeast and the tyrosine-specific system from Escherichia coli.

The kinetics of the interaction of tRNASer and seryl-tRNA synthetase from yeast as well as of tRNATyr and tyrosyl-tRNA synthetase from Escherichia coli have been investigated by temperature-jump experiments. It could be shown that complex formation proceeds in two distinct steps. This was demonstrated for both the first and the second binding site. The two-step mechanism was deduced from the characteristic concentration dependence of the relaxation times. Seryl-tRNA synthetase recombines with the first tRNA to form an intermediate complex (kI12, kI21), which is transformed in a fast reaction to the final 1:1 complex (kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 X 10(8) M-1 S-1; kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 x 10(8) M-1 S-1; kI21 = 220 S-1; kI23 = 760 S-1; kI32 = 330 S-1. The 1:1 complex can bind a second tRNA. At pH 7.2 without added salt the rate constants are: KII2 = 0.9 X 10(8) M-1 S-1; kII21 = 270 S-1; kII23 = 120 S-1; kII32 = 1250 S-1. The tyrosine-specific system behaves very similarly to the serine-specific system. Data are given for pH 7.2 (pH 6.0) for the binding of the second tRNA: kII12 = 1 X 10(8) (2.5 X 10(8)) M-1 S-1; kII21 = 470 (170) S-1; kII23 = 150 (530) S-1; kII32 = 1540 (720) S-1. The kinetic results are discussed in terms of their relevance to the recognition process and their relation to the anticooperative binding behaviour of tRNA to synthetase.

Amino Acyl-tRNA Synthetases↗