The effect of tRNA and tryptophanyl adenylate on limited proteolysis of beef pancreas tryptophanyl-tRNA synthetase.

Limited proteolysis of tryptophanyl-tRNA synthetase was used to detect changes in the enzyme molecule in the presence of substrates. Trypsinolysis of each of the two identical subunits occurs in succession from the N-terminus as follows: 60 leads to 51 leads to 40 leads to 24 kilodaltons. The transition 51 leads to 40 is hindered in tryptophanyl adenylate.enzyme complex. Yeast tRNATrp accelerates the first steps of hydrolysis and decelerates the transition 40 leads to 24. Once tRNATrp is added to the synthetase.adenylate complex, the protective effect of the adenylate disappears. The same effects are found also in the presence of tRNATrp oxidized with NaI04 and tRNATrp lacking the 3'-terminal adenosine. Oxidized tRNATrp (but not tRNATrp without the 3'-A) accelerates tryptophan-dependent hydrolysis of ATP catalyzed by the enzyme. A scheme is proposed for the interaction of yeast tRNATrp with beef pancreas tryptophanyl-tRNA synthetase involving the association of tRNA with a positively charged site(s) of the enzyme and the changes in the conformation of enzyme manifesting itself in unfolding of the acidic N-terminal fragment of the polypeptide chain and in the exposure of the adenylate.     

Initial position of aminoacylation of individual Escherichia coli, yeast, and calf liver transfer RNAs.

Transfer RNAs from Escherichia coli, yeast (Sacharomyces cerevisiae), and calf liver were subjected to controlled hydrolysis with venom exonuclease to remove 3'-terminal nucleotides, and then reconstructed successively with cytosine triphosphate (CTP) and 2'- or 3'-deoxyadenosine 5'-triphosphate in the presence of yeast CTP(ATP):tRNA nucleotidyltransferase. The modified tRNAs were purified by chromatography on DBAE-cellulose or acetylated DBAE-cellulose and then utilized in tRNA aminoacylation experiments in the presence of the homologous aminoacyl-tRNA synthetase activities. The E. coli, yeast, and calf liver aminoacyl-tRNA synthetases specific for alanine, glycine, histidine, lysine, serine, and threonine, as well as the E. coli and yeast prolyl-tRNA synthetases and the yeast glutaminyl-tRNA synthetase utilized only those homologous modified tRNAs terminating in 2'-deoxyadenosine (i.e., having an available 3'-OH group). This is interpreted as evidence that these aminoacyl-tRNA synthetases normally aminoacylate their unmodified cognate tRNAs on the 3'-OH group. The aminoacyl-tRNA synthetases from all three sources specific argining, isoleucine, leucine, phenylalanine, and valine, as well as the E. coli and yeast enzymes specific for methionine and the E. coli glutamyl-tRNA synthetase, used as substrates exclusively those tRNAs terminating in 3'-deoxyadenosine. Certain aminoacyl-tRNA synthetases, including the E. coli, yeast, and calf liver asparagine and tyrosine activating enzymes, the E. coli and yeast cysteinyl-tRNA synthetases, and the aspartyl-tRNA synthetase from yeast, utilized both isomeric tRNAs as substrates, although generally not at the same rate. While the calf liver aspartyl- and cysteinyl-tRNA synthetases utilized only the corresponding modified tRNA species terminating in 2'-deoxyadenosine, the use of a more concentrated enzyme preparation might well result in aminoacylation of the isomeric species. The one tRNA for which positional specificity does seem to have changed during evolution is tryptophan, whose E. coli aminoacyl-tRNA synthetase utilized predominantly the cognate tRNA terminating in 3'-deoxyadenosine, while the corresponding yeast and calf liver enzymes were found to utilize predominantly the isomeric tRNAs terminating in 2'-deoxyadenosine. The data presented indicate that while there is considerable diversity in the initial position of aminoacylation of individual tRNA isoacceptors derived from a single source, positional specificity has generally been conserved during the evolution from a prokaryotic to mammalian organism.     

Binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase from bovine pancreas under pH conditions of maximum activity. Analysis by ultracentrifugation, fluorescence quenching and chemical modification

The binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef is examined by three approaches, under pH conditions of maximum activity (pH 8.0). (1) Analytical ultracentrifugation evidences the binding of a single mol of tRNATrp in a 2.5-10 microM concentration range. (2) tRNATrp quenches the fluorescence of the enzyme. The dependence of this fluorescence quenching on the tRNATrp concentration (0.1-4 microM) reflects also the binding of 1 mol of tRNA per mol of enzyme, with a Kd value of 0.19 +/- 0.02 microM. (3) tRNATrp protects the enzyme against derivatization by oxidized ATP. Out of the two fast-reacting lysine residues of the native enzyme, only one is prevented from reacting by tRNATrp in the 0.5-110 microM concentration range. This protection can be significantly analyzed only by assuming a one-to-one complex between the enzyme and tRNA. These results, obtained at pH 8.0 and 25 degrees C, are in contrast with the stoichiometry of 2 mol of tRNA to 1 mol of enzyme, previously observed at pH 6.0 and 4 degrees C.     

Reaction of tryptophanyl-tRNA synthetase from beef pancreas with periodate-oxidized ATP

Tryptophanyl-tRNA synthetase from beef pancreas reacts with periodate-oxidized ATP according to biphasic kinetics. A rapid phase involves two groups of the protein, presumably lysine side-chains. The slow phase corresponds to the reaction of a larger number of groups. The time-course of the partial losses of the ATP-PPi isotopic exchange and of the aminoacylation activities of the enzyme follow the labelling of the two fast-reacting groups. However, the ability of the enzyme to form a bis(tryptophanyladenylate)-enzyme complex is not lost after reaction of these two groups with the reagent. The affinity for ATP is also unaffected by this initial labelling of the protein, as seen from the Km values of this substrate in the ATP-PPi isotopic exchange reaction. These data suggest that, in this fast initial reaction, oxidized ATP reacts neither with specific ATP-binding groups of the enzyme nor with any major catalytic residue of the tryptophan-activation site. In contrast with this first step, the further slow labelling of lysine residues leads to a disappearance of the aminoacylation ability of the enzyme, while it does not further affect the ATP-PPi exchange activity. The behaviour of beef tryptophanyl-tRNA synthetase during derivatization with oxidized ATP is therefore at variance with that which has been described for the homologous E. coli enzyme.     

Isoleucyl-tRNA synthetase from Escherichia coli MRE 600. Different pathways of the aminoacylation reaction depending on presence of pyrophosphatase, order of substrate addition in the pyrophosphate exchange, and substrate specificity with regard to ATP analogs

The substrate specificity of isoleucyl-tRNA synthetase from Escherichia coli MRE 600 with regard to ATP analogs has been compared with the results obtained with isoleucyl-tRNA synthetase from yeast. The enzyme from E. coli is less specific, the two enzymes exhibit different topographies of their active centres. The order of substrate addition to isoleucyl-tRNA synthetase from E. coli MRE 600 has been investigated by bisubstrate kinetics, product inhibition and inhibition by substrate analogs. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. As found for isoleucyl-tRNA synthetase from yeast, the results of the pyrophosphate exchange studies indicate the possibility of formation of E . Ile-AMP . ATP complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, release of pyrophosphate and subsequent addition of a second molecule of ATP. For the aminoacylation in the absence of pyrophosphatase, a rapid-equilibrium random ter addition of the substrates is found whereas the enzyme from yeast exhibits a steady-state ordered ter-ter mechanism; in the presence of pyrophosphatase the mechanism is bi-uni uni-bi ping-pong similarly as observed for the yeast enzyme. A comparison of inhibition patterns obtained with N(6)-benzyladenosine 5'-triphosphate under different assay conditions (spermine or magnesium ions, addition of pyrophosphatase) indicates that even more than two pathways of the aminoacylation may exist. The catalytic cycles of the two mechanisms derived from the observed orders of substrate addition and product release include the same enzyme substrate complex (E . tRNA . Ile-AMP) for the aminoacyl transfer reaction. The kcat values, however, are considerably different: kcat of the sequential pathway is about 40% lower than kcat of the ping-pong mechanism.     

Evidence for a conserved relationship between an acceptor stem and a tRNA for aminoacylation.

The anticodon-independent aminoacylation of RNA hairpin helices that reconstruct tRNA acceptor stems has been demonstrated for at least 10 aminoacyl-tRNA synthetases. For Escherichia coli cysteine tRNA synthetase, the specificity of aminoacylation of the acceptor stem is determined by the U73 nucleotide adjacent to the amino acid attachment site. Because U73 is present in all known cysteine tRNAs, we investigated the ability of the E. coli cystein enzyme to aminoacylate a heterologous acceptor stem. We show here that a minihelixCys based on the acceptor-T psi C stem of yeast tRNACys is a substrate for the E. coli enzyme, and that aminoacylation of this minihelix is dependent on U73. Additionally, we identify two base pairs in the acceptor stem that quantitatively convert the E. coli acceptor stem to the yeast acceptor stem. The influence of U73 and these two base pairs is completely retained in the full-length tRNA. This suggests a conserved relationship between the acceptor stem alone and the acceptor stem in the context of a tRNA for aminoacylation with cysteine. However, the primary determinant in the species-specific aminoacylation of the E. coli and yeast cysteine tRNAs is a tertiary base pair at position 15:48 outside of the acceptor stem. Although E. coli tRNACys has an unusual G15:G48 tertiary base pair, yeast tRNACys has a more common G15:C48 that prevents efficient aminoacylation of yeast tRNACys by the E. coli enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic evidence for half-of-the-sites reactivity in tRNATrp aminoacylation by tryptophanyl-tRNA synthetase from beef pancreas

The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flow method. The transfer of tryptophan to tRNATrp was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-[14C]tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-[14C]tryptophan were used. We measured either the formation of a single labeled aminoacyl-tRNATrp per enzyme subunit or the turnover of labeled aminoacyl-tRNATrp synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute "half-of-the-sites reactivity"); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNATrp under each labeling condition were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate of aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process.     

Evidence that a major determinant for the identity of a transfer RNA is conserved in evolution

We observed recently that a single G3.U70 base pair in the amino acid acceptor stem of an Escherichia coli alanine tRNA is a major determinant for its identity. Inspection of tRNA sequences shows that G3.U70 is unique to alanine in E. coli and is present in eucaryotic cytoplasmic alanine tRNAs. We show here that single nucleotide changes of G3.U70 to A3.U70 or to G3.C70 eliminate in vitro aminoacylation of an insect and of a human alanine tRNA by the respective homologous synthetase. Compared to the influence of G3.U70, other sequence variations in tRNAAla have a relatively small effect on aminoacylation by the insect and human enzymes. In addition, while these eucaryotic tRNAs have nucleotide differences from E. coli alanine tRNA, they are heterologously charged only with alanine when expressed in E. coli. The results indicate a functional role for G3.U70 that is conserved in evolution. They also suggest that the sequence differences between E. coli and the eucaryotic alanine tRNAs at sites other than the conserved G3.U70 do not create major determinants for recognition by any other bacterial enzyme.     

A factor affecting stimulation of aminocylation in plants.

The presence of a factor stimulating the reaction of aminoacylation tRNAs was found in the seeds of lupin, with a molecular weight of 950 as estimated by gel filtration. The influence of this factor on the kinetics of the aminoacylation reactions of lupin, Escherichia coli, Baker's yeast and heterogeneous systems was investigated. This factor inhibits the esterification reaction of aminoacyl-tRNA synthetases from bacteria and yeast. Its influence on the optimum pH activity of isoleucyl-tRNA synthetase from lupin was determined.     

On the roles of magnesium and spermidine in the isoleucyl-tRNA synthetase reaction. Analysis of the reaction mechanism by total rate equations

The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PPi exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine.ATP complex. Spermidine.ATP is a weaker substrate. The role of spermidine.ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines.     

Mutual conformational changes of tryptophanyl-tRNA synthetase and tRNATrp in the course of their specific interaction

tRNATrp (beef, yeast) is capable of accelerating limited tryptic hydrolysis of the N-terminal part in the polypeptide chains of dimeric beef pancreas tryptophanyl-tRNA synthetase; it can also eliminate the protective effect of tryptophanyl adenylate on the enzyme proteolysis. The effect of tRNA on the proteolysis is manifested even when the 3'-CCA terminus is removed. It has been concluded that the conformation of the synthetase changes when it forms a complex with tRNATrp. Yeast tRNATrp lacking the 3'-half of the acceptor stem can still interact with the synthetase and, to certain extent, induces changes in the conformation of the latter. The susceptibility of single-stranded and double-stranded regions of tRNATrp to cleavage with endonucleases has been studied, and the results are indicative of the fact that, regardless of considerable differences in the nucleotide sequence of yeast and beef tRNATrp, their three-dimensional structures are similar. This fact is consistent with the finding that parameters for the interaction of these tRNAsTrp with beef tryptophanyl-tRNA synthetase are rather close. The three-dimensional structure of tRNATrp is altered when the enzyme forms a complex with it, as seen from (a) a change in the circular dichroic spectrum and (b) an elevated susceptibility of the anticodon and, apparently, acceptor stems to cleavage with nuclease. The conversion of exposed cytidine residues in tRNATrp into uridine residues results in a loss of the acceptor activity; the capability to accelerate limited tryptic hydrolysis of tryptophanyl-tRNA synthetase is also lost although the enzyme-substrate complex, as seen from circular dichroic spectra, can still be formed. The conversion of cytosine in the anticodon stem into uracil modifies the conformation of the anticodon stem. The anticodon arm (including the anticodon) and the acceptor stem play an essential role in the interaction between tRNATrp and tryptophanyl-tRNA synthetase.     

Recognition of individual procaryotic and eucaryotic transfer-ribonucleic acids by B subtilis adenine-1-methyltransferase specific for the dihydrouridine loop.

Bulk tRNA from yeast and Rat liver can be methylated in vitro with -adenosylmethionine and B, subtilis extracts. The sole product formed is 1-methyladenosine (m1A). This tRNA (adenine-1) methyltransferase converts quantitatively the 3'-terminal adenosine-residue in the dihydrouridine-loop of tRNAThr and tRNATyr from yeast into m1A. Out of 16 eucaryotic tRNAs with known sequences 6 accepted methyl groups, all at a molar ratio of 1. These tRNAs have in common an unpaired adenosine-residue at the specific site in the sequence Py-A-A+-G-G-C-m2G. Out of 12 tRNAs from E. coli 6 served as specific substrates. These E. coli tRNAs also have an unpaired adenosine-residue at the 3'-end of the D-loop. Besides restrictions in primary structure intact secondary and tertiary structure is important for recognition of the specific tRNAs by the enzyme.     

Response of specific transfer-ribonucleic-acid levels to amino-acid deprivation in Friend leukemia cells.

In eukaryotes, the levels of specific tRNAs are closely correlated with the demands for their cognate amino acids in protein synthesis. To account for this phenomenon, we have proposed that the extent of aminoacylation of a given tRNA species in vivo controls the relative rate of synthesis or turnover of that species. Previously, we reported that Friend leukemia cells respond to histidine deprivation by increasing their relative level of tRNAHis by as much as two-fold, with no change in the relative level of tRNALeu. In this paper, we show that deprivation of leucine or tryptophan also causes a specific increase in the relative level of tRNAs cognate to the deprived amino acid. At least in the case of tRNATrp, the increases in relative tRNA levels are preceded by extensive declines in the steady-state extent of aminoacylation of the tRNA in vitro. We also find that different isoacceptors may respond differently to amino acid deprivation. These results suggest that decreased extents of aminoacylation of a given tRNA species in vivo cause increases in the relative rate of synthesis or decreases in the relative rate of degradation of that species.     

Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl-tRNA synthetase

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl-tRNA synthetase. The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and T psi arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPhe since beef tRNATrp showed a more stable D stem and a less stable T psi stem. When alkylation by ethylnitrosourea was studied with the tRNATrp X tryptophanyl-tRNA synthetase complex we found that phosphates located at the 5' side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N-3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase X tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl-tRNA synthetase was confirmed in nuclease S1 mapping experiments.     

The mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena are indistinguishable.

The mitochondrial and cytoplasmic valyl tRNA synthetases from Tetrahymena pyriformis are indistinguishable. These synthetases cannot be differentiated through hydroxylapatite, DEAE-cellulose, or phosphocellulose column chromatography. Both enzymes show the same mean sedimentation coefficient of 5.9 S in sucrose gradient centrifugation analysis; when bound with tRNA, they are relatively stable and sediment at 7.8 S. The temperature optimum for aminoacylation reaction is 27.5 °C, the optimum Mg²⁺ concentration is 4.4 mm, and substrate affinities (K_m values) for valine and ATP in aminoacylation are the same for both enzymes at 1.0 μm and 2.5 mm, respectively. These enzymes show identical specificities for acylation of different tRNA species, i.e., Tetrahymena and rat liver tRNAs can be equally well recognized, but no significant acylation can be observed with Escherichia coli and Saccharomyces tRNAs. These observations suggest the probable molecular identity of mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena.     

pH profiles and isotope effects for aconitases from Saccharomycopsis lipolytica, beef heart, and beef liver. alpha-Methyl-cis-aconitate and threo-Ds-alpha-methylisocitrate as substrates

alpha-Methyl-cis-aconitate (cis-2-butene-1,2,3-tricarboxylate) was converted only to alpha-methylisocitrate (3-hydroxybutane-1,2,3-tricarboxylate) by aconitases from beef liver or S. lipolytica. While the kinetic parameters of beef liver (cytoplasmic) or heart (mitochondrial) aconitases did not vary over the pH range 4.9-9 with the natural substrates, and only slightly with the alpha-methyl substrates, the yeast aconitase exhibited a bell-shaped pH profile with all substrates and for binding of the competitive inhibitor, tricarballylate, with pK values around 7 and 9. The third pK of the substrates does not affect V/K, showing that these pK's are for catalytic groups on the enzyme. One of these catalytic groups presumably removes a proton to give the carbanion intermediate in the reaction, and the other protonates the hydroxyl group when it is eliminated to give water, possibly with the assistance of the Fe-S center. Beef liver aconitase showed a primary deuterium isotope effect of 1.12 (measured by equilibrium perturbation with deuterated alpha-methylisocitrate) which was pH independent and only slightly greater than the equilibrium isotope effect. Isotope effects with the yeast enzyme were also pH independent but about 1.22 on V/K (or when measured by equilibrium perturbation) and 1.7 on V. These data suggest a kinetic mechanism for beef aconitases in which product release occurs only by displacement by the substrate in a step independent of pH or of the protonation state of the substrate. With the yeast enzyme, product displacement either depends on the protonation state of the catalytic groups on the enzyme or can occur spontaneously at a finite rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete purification and studies on the structural and kinetic properties of two forms of yeast valyl-tRNA synthetase.

Two forms of baker's yease valyl-tRNA synthetase have been purified to apparent homogeneity by classical methods. It was demonstrated that one of the two forms of the enzyme originates from the other by proteolysis, the respective amounts of each form depending on the physiological state of the yeast. The species mainly isolated from exponential growing yeast cells is a monomer of 130,000 daltons molecular weight. In stationary phase cells or in commercial yeast the major species is a degraded monomer of 120,000 daltons molecular weight ; however when the purification is carried out in the presence of phenylmethyl-sulphonyl fluoride, or diisopropylfluorophosphate large amounts of the not - degreded monomer can be obtained. Of great practical usefulness is the fact that large amounts of the native enzyme can be obtained pure after only two chromatographic steps on DEAE-cellulose and hydroxylapatite. The kinetic constants for valine, ATP and tRNAVal were determined, as well as the optimum aminoacylation conditions. It was found that the specific activity of the nondegraded valyl-tRNA synthetase is higher than that of the proteolysed enzyme for the aminoacylation reaction. On the contrary, both forms have the same ATP-pyroposphate exchange activity. The amino acids composition of the native enzyme was established. The tryptic fingerprints of the two valyl-tRNA synthetases were studied. Essentially similar maps were obtained. The number of the spots in the fingerprints indicates that the enzymes contain a high proportion of repeated sequences.     

ATP-induced activation of the aminoacylation of tRNA by the isoleucyl-tRNA synthetase from Escherichia coli

The rate of aminoacylation of tRNA catalyzed by the isoleucyl-tRNA synthetase form Escherichia coli has been measured. A steady-state kinetic analysis of the rate as a function of the concentration of ATP gave nonlinear Hanes plots. ATP behaves as an activator of the reaction. The activation is observed at a low magnesium ion concentration and in the presence of spermidine. The presence of inorganic pyrophosphate or AMP enhances the activation. The results are consistent with a mechanism in which the binding of a second molecule of ATP increases the rate of dissociation of Ile-tRNA from the enzyme.