Phenylalanine transfer ribonucleic acid synthetase from Drosophila melanogaster: Purification and properties

Phenylalanine transfer ribonucleic acid synthetase from Drosophila melanogaster has been purified 1400-fold over a crude 230,000g supernatant fraction. The optimum activity of the enzyme occurs at magnesium concentrations above 10 mm at 37 °C and pH 7.5. At a 50 mm Mg²⁺ concentration, NH₄⁺ stimulates the ATP-PP₁ exchange reaction as much as 2-fold. Ammonium chloride causes an increase in the V with no change in the K_m with phenylalanine as substrate. Homologous (Drosophila) tRNA, in the presence of NH₄⁺, further stimulates the ATP-PP_i, exchange reaction but inhibits the reaction in the absence of NH₄⁺.In the presence of its substrates the enzyme is inactivated by NEM to varying degrees depending upon the substrate or combinations of substrates used. In the presence of phenylalanine the enzyme is partially protected but both ATP and tRNA make the enzyme more susceptible to inactivation. NEM together with ATP and tRNA or all three substrates results in near-total inactivation.     

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme.Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α₂ An ATP analogue, γ-(p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA^Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K₁ = 1 × 10^?3m (K_mfor ATP = 2 × 10^?4m). The covalent photoaddition of azido-ATP³ results in complete loss of enzymatic activity in both the ATP-[³²P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme.Two binding sites for tRNA^Trp have been found from complex formation at pH 5.8 in the presence of Mg²⁺. The two tRNA molecules bind, with K_dis = 3.6 × 10^?8m and K_dis = 0.9 × 10^?6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA.N-chlorambucilyl-tryptophanyl-tRNA^Trp and TRSase form a complex with K_dis = 5.5 × 10^?8m at pH 5.8 in the presence of 10 mm-Mg²⁺. This value is similar to the value of K_dis for tryptophanyl-tRNA of 4.8 × 10^?8m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA^Trp whereas the tRNA devoid of tRNA^Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected.Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [³²P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA^Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[³²P]pyrophosphate exchange and aminoacylation activity.The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein.Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.     

The tRNA Recognition Mechanism of Folate/FAD-dependent tRNA Methyltransferase (TrmFO)

The conserved U54 in tRNA is often modified to 5-methyluridine (m⁵U) and forms a reverse Hoogsteen base pair with A58 that stabilizes the L-shaped tRNA structure. In Gram-positive and some Gram-negative eubacteria, m⁵U54 is produced by folate/FAD-dependent tRNA (m⁵U54) methyltransferase (TrmFO). TrmFO utilizes N⁵,N¹⁰-methylenetetrahydrofolate (CH₂THF) as a methyl donor. We previously reported an in vitro TrmFO assay system, in which unstable [¹⁴C]CH₂THF was supplied from [¹⁴C]serine and tetrahydrofolate by serine hydroxymethyltransferase. In the current study, we have improved the TrmFO assay system by optimization of enzyme and substrate concentrations and introduction of a filter assay system. Using this assay, we have focused on the tRNA recognition mechanism of TrmFO. 42 tRNA mutant variants were prepared, and experiments with truncated tRNA and microhelix RNAs revealed that the minimum requirement of TrmFO exists in the T-arm structure. The positive determinants for TrmFO were found to be the U54U55C56 sequence and G53-C61 base pair. The gel mobility shift assay and fluorescence quenching showed that the affinity of TrmFO for tRNA in the initial binding process is weak. The inhibition experiments showed that the methylated tRNA is released before the structural change process. Furthermore, we found that A38 prevents incorrect methylation of U32 in the anticodon loop. Moreover, the m¹A58 modification clearly accelerates the TrmFO reaction, suggesting a synergistic effect of the m⁵U54, m¹A58, and s²U54 modifications on m⁵s²U54 formation in Thermus thermophilus cells. The docking model of TrmFO and the T-arm showed that the G53-C61 base pair is not able to directly contact the enzyme.     

Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase

In most organisms, the widely conserved 1-methyl-adenosine58 (m¹A₅₈) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m¹A₅₇ being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (_PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. _PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A₅₇ but not A₅₈, confirming that _PabTrmI methylates efficiently the first adenine of the A₅₇A₅₈A₅₉ sequence. _PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m¹A₅₈ formation triggers RNA release. A model of the protein–tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.     

Trace 5-Methylaminomethyl-2-selenouridine in bovine tRNA and the selenouridine synthase activity in bovine liver

We measured the amount of Se in bovine liver tRNA. tRNA was chromatographed on a BD-cellulose column and Se-rich tRNA was eluted from the column in front of a main tRNA peak. There was 0.3 mmol Se/mol of tRNA and this level is about one tenth that of Escherichia coli tRNA. This suggests the presence of an enzyme that modifies tRNA with Se in bovine liver. We isolated the activity of this enzyme (selenouridine synthase) by chromatography of bovine liver extracts on a DEAE-cellulose column. ATP and selenophosphate synthetase, as well as selenouridine synthase and tRNA, were necessary for the reaction. ⁷⁵Se was used to label the reaction products, which were analyzed by TLC after digestion with ribonuclease T₂. The position of the ⁷⁵ Se-nucleotide on a TLC plate was identical to that of the Se-nucleotide, 5-methylaminomethyl-2-seleno-Up, prepared from ⁷⁵Se-tRNA in E. coli.     

Affinity labeling of phenylalanyl-tRNA synthetase from E.coli MRE-600 by E.coli tRNAphe containing photoreactive group

The photoinduced reaction of phenylalanyl-tRNA synthetase (E.C. 6.1.1.20) from E.coli MRE-600 with tRNA^phe containing photoreative p-N₃-C₆H₄-NHCOCH₂-group attached to 4-thiouridine sU₈ (azido-tRNA^phe) was investigated. The attachment of this group does not influence the dissociation constant of the complex of Phe-tRNA^phe with the enzyme,however it results in sevenfold increase of K_m in the enzymatic aminoacylation of tRNA^phe. Under irradiation at 300 nm at pH 5.8 the covalent binding of [¹⁴C]-Phe-azido-tRNA^phe to the enzyme takes place 0.3 moles of the reagent being attached per mole of the enzyme. tRNA prevents the reaction. Phenylalanine, ATP,ADP,AMP, adenosine and pyrophosphate (2.5 × x 10⁻³ M) don't affect neither the stability of the tRNA-enzyme complex nor the rate of the affinity labelling. The presence of the mixture of either phenylalanine or phenylalaninol with ATP as well as phenylalaninol adenylate exibits 50% inhibition of the photoinduced reaction. Therefore, the reaction of [¹⁴C]-Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in ¹. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNA - enzyme interaction.     

Neutron scattering studies of escherichia coli tyrosyl-trna synthetase and of its interaction with trna tyr

Small-angle neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase indicate that in solution this enzyme is a dimer of M_r, 91 (±6) × 10³ with a radius of gyration R_G of 37.8 ± 1.1 Å.The increase in the scattering mass of the enzyme upon binding tRNA^Tyr has been followed in 20 mm-imidazole · HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol, 150 mm-KCl. A stoichiometry of one bound tRNA per dimeric enzyme molecule was found. The R_G of the complex is equal to 41 ± 1 Å. Titration experiments in 74% ²H₂O, close to the matching point of tRNA, show an R_G of 38.5 ± 1 Å for the enzyme moiety in the complex. From these values, a minimum distance of 49 Å between the centre of mass of the bound tRNA and that of the enzyme was calculated.In low ionic strength conditions (20 mm-imidazole-HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol) and at limiting tRNA concentrations with respect to the enzyme, titrations of the enzyme by tRNA^Tyr are characterized by the appearance of aggregates, with a maximum scattered intensity at a stoichiometry of one tRNA per two enzyme molecules. At this point, the measured M_r and R_G values are compatible with a compact 1:2, tRNA: enzyme complex. This complex forms with a remarkably high stability constant: (enzyme:tRNA:enzyme)/(enzyme:tRNA)(enzyme) of 0.1 to 0.3(× 10⁶) m^?1 (at 20 °C). Upon addition of more tRNA, the complex dissociates in favour of the 1:1, enzyme:tRNA complex, which has a higher stability constant (1 to 3 (× 10⁶) m^?1).     

Antico-operative binding of initiator transfer RNAMet to methionyl-transfer RNA synthetase from Escherichia coli: neutron scattering studies.

The interaction of methionyl-tRNA synthetase with initiator tRNA^Met has been investigated by neutron scattering. On the basis of parallel fluorescence measurements, two types of titrations have been performed. (1) In the presence of 10 mm-MgCl₂, a condition which insures antico-operative binding of two tRNA molecules to the enzyme dimer. (2) With saturating amounts of 5′-AMP and l-methioninol, in the presence of 50 mm-MgCl₂, conditions which allow two transfer RNA molecules to bind the dimer with very similar affinities.Varying the solvent density (²H₂O fraction) in the samples has allowed the identification by neutron scattering of changes in the radius of gyration and in the degree of dissociation of the enzyme dimer upon tRNA binding. In buffer containing 10 mm-MgCl₂, at each contrast studied, the binding process involves two steps. Firstly, one tRNA^met_f molecule binds easily to one dimeric enzyme molecule with an associated decrease of the radius of gyration of the enzyme moiety. The centre of mass of this tRNA lies very close to the centre of mass of the protomer with which it associates. Then, at higher tRNA concentration, a second tRNA molecule binds to the enzyme. However, the affinity of this second site is very much weaker. With the binding of the second tRNA, the radius of gyration of the enzyme moiety increases markedly. Concomitant limited dissociation of the dimer is suggested by the experimental data. These observations combined with the fact that, in 50 mm-MgCl₂ both the increased radius of gyration and the partial dissociation of the enzyme are accomplished in the absence of tRNA and remain unaffected upon binding one or two tRNA, confirm that the hindrance to binding a second tRNA in 10 mm-MgCl₂ arises from the constrained conformation of the one tRNA-enzyme complex.     

Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis

tRNA (Ura-5-)methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the 5-carbon of a specific Urd residue in tRNA. This results in stoichiometric release of tritium from [5-3H]Urd-labeled substrate tRNA isolated from methyltransferase-deficient Escherichia coli. The enzyme also catalyzes an AdoMet-independent exchange reaction between [5-3H]-Urd-labeled substrate tRNA and protons of water at a rate that is about 1% that of the normal methylation reaction, but with identical stoichiometry. S-Adenosylhomocysteine inhibits the rate of the exchange reaction by 2-3-fold, whereas an analogue having the sulfur of AdoMet replaced by nitrogen accelerates the exchange reaction 9-fold. In the presence (but not absence) of AdoMet, 5-fluorouracil-substituted tRNA (FUra-tRNA) leads to the first-order inactivation of the enzyme. This is accompanied by the formation of a stable covalent complex containing the enzyme, FUra-tRNA, and the methyl group of AdoMet. A mechanism for catalysis is proposed that explains both the 5-H exchange reaction and the inhibition by FUra-tRNA: the enzyme forms a covalent Michael adduct with substrate or inhibitor tRNA by attack of a nucleophilic group of the enzyme at carbon 6 of the pyrimidine residue to be modified. As a result, an anion equivalent is generated at carbon 5 that is sufficiently reactive to be methylated by AdoMet. Preliminary experiments and precedents suggest that the nucleophilic catalyst of the enzyme is a thiol group of cysteine. The potent irreversible inhibition by FUra-tRNA suggests that a mechanism for the "RNA" effects of FUra may also involve irreversible inhibition of RNA-modifying enzymes.     

Antico-operative binding of bacterial and mammalian initiator tRNAMet to methionyl-tRNA synthetase from Escherichia coli

The reaction scheme of methionyl-tRNA synthetase from Escherichia coli with the initiator tRNA_s^Met from E. coli and rabbit liver, respectively, has been resolved. The statistical rate constants for the formation, k_R, and for the dissociation, k_D, of the 1:1 complex of these tRNAs with the dimeric enzyme have been calculated. Identical k_R values of 250 μm^?1 s^?1 reflect similar behaviour for antico-operative binding of both tRNA_s^Met to native methionyl-tRNA synthetase. Advantage was taken of the difference in extent of tryptophan fluorescence-quenching induced by the bacterial and mammalian initiator tRNA_s^Met to measure the mode of exchange of these tRNAs antico-operatively bound to the enzyme. Analysis of the results reveals that antico-operativity does not arise from structural asymmetric assembly of the enzyme subunits. Indeed, both subunits can potentially bind a tRNA molecule. Exchange between tRNA molecules can occur via a transient complex in which both sites are occupied. Either strong and weak sites reciprocate between subunits on the transient complex or occupation of the weak site induces symmetry of this complex. While in the present case, these two alternatives are kinetically indistinguishable, they do account for the observation that, upon increasing the concentration of the competing mammalian tRNA, the rate of exchange of the E. coli initiator tRNA^Met is enhanced, due to its faster rate of dissociation from the transient complex. Finally, it has been verified that in the case of the trypsin-modified methionyl-tRNA synthetase which cannot provide more than one binding site for tRNA, exchange of enzymebound bacterial tRNA by mammalian tRNA does proceed to a limiting rate independent of the mammalian tRNA concentration present in the solution.     

Transfer RNA determinants for translational editing by Escherichia coli valyl-tRNA synthetase

Valyl-tRNA synthetase (ValRS) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threonine. To minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate tRNA^Val. Editing occurs at a site functionally distinct from the aminoacylation site of ValRS and previous results have shown that the 3′-terminus of tRNA^Val is recognized differently at the two sites. Here, we extend these studies by comparing the contribution of aminoacylation identity determinants to productive recognition of tRNA^Val at the aminoacylation and editing sites, and by probing tRNA^Val for editing determinants that are distinct from those required for aminoacylation. Mutational analysis of Escherichia coli tRNA^Val and identity switch experiments with non-cognate tRNAs reveal a direct relationship between the ability of a tRNA to be aminoacylated and its ability to stimulate the editing activity of ValRS. This suggests that at least a majority of editing by the enzyme entails prior charging of tRNA and that misacylated tRNA is a transient intermediate in the editing reaction.     

N2-guanine specific transfer RNA methyltransferase II from rat liver

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N²-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m²G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA^Arg, tRNA^Phe, and tRNA^Val yielded significantly more m²G than the bulk tRNA. The K_m for tRNA^Arg in the methylation reaction with enzymes from either tissue was 7.8 × 10⁻⁷ M as compared to the value 1 × 10⁻⁵ M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA^Arg, tRNA^Phe, and tRNA^Val, A-m²G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5′-end contained in a sequence (s⁴)U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

Mature methyl-deficient tRNA isolated from a mammary adenocarcinoma

A transplantable rat tumor, mammary adenocarcinoma 13762, accumulates tRNA which can be methylated in vitro by mammalian tRNA (adenine-1) methyltransferase. This unusual ability of the tumor RNA to serve as substrate for a homologous tRNA methylating enzyme is correlated with unusually low levels of the A₅₈-specific adenine-1 methyltransferase. The nature of the methyl-accepting RNA has been examined by separating tumor tRNA on two-dimensional polyacrylamide gels. Comparisons of ethidium bromide-stained gels of tumor vs. liver tRNA show no significant quantitative differences and no accumulation of novel tRNAs or precursor tRNAs in adenocarcinoma RNA. Two-dimensional separations of tumor RNA after in vitro [¹⁴C]methylation using purified adenine-1 methyltransferase indicate that about 25% of the tRNA species are strongly methyl-accepting RNAs. Identification of six of the tRNAs separated on two-dimensional gels has been carried out by hybridization of cloned tRNA genes to Northern blots. Three of these, tRNA^Lys3, tRNA^Gln and tRNA^Met_i, are among the adenocarcinoma methyl-accepting RNAs. The other three RNAs, all of which are leucine-specific tRNAs, show no methyl-accepting properties. Our results suggest that low levels of a tRNA methyltransferase in the adenocarcinoma cause selected species of tRNA to escape the normal A₅₈ methylation, resulting in the appearance of several mature tRNAs which are deficient in 1-methyladenine. The methyl-accepting tRNAs from the tumor appear as ethidium bromide-stained spots of similar intensity to those seen for RNA from rat liver; therefore, methyladenine deficiency does not seem to impair processing of these tRNAs.     

Studies of odd bases in yeast mitochondrial tRNA: III. Characterization of the tRNA methylases associated with the mitochondria

Whereas m¹G, m²G, m₂²G, m⁷G, T, m¹A, m⁵C and Cm methylase activities were found in total cell enzyme of $\text{Saccharomyces cerevisiae}$ using under-methylated $\text{E. coli}$ tRNA and $\text{E. coli}$ B tRNA in reaction with or without Mg⁺⁺, only m¹G, m²G, m₂²G and T methylases occurred in mitochondria. Mitochondrial and cytoplasmic tRNA cannot be methylated by their homologous enzymes; only mitochondrial tRNA can be methylated in a heterologous reaction by total cell enzyme with formation of T, m⁵C, m¹A and low amounts of m²G and m₂²G.     

Determinants in tRNA for activation of arginyl-tRNA synthetase: evidence that tRNA flexibility is required for the induced-fit mechanism

Arginyl-tRNA synthetase (ArgRS) catalyzes formation of arginyl-adenylate in a tRNA-dependent reaction. Previous studies have revealed that conformational changes occur upon tRNA binding. In this study, we analyzed the sequence and structural features of tRNA that are essential to activate the catalytic center of mammalian arginyl-tRNA synthetase. Here, tRNA variants with different activator potential are presented. The three regions that are crucial for activation of ArgRS are the terminal adenosine, the D-loop, and the anticodon stem-loop of tRNA. The Add-1 N-terminal domain of ArgRS, which has the very unique property among aminoacyl-tRNA synthetases to interact with the D-loop in the corner of the convex side of tRNA, has an essential role in anchoring tRNA and participating in tRNA-induced amino acid activation. The results suggest that locking the acceptor extremity, the anticodon loop, and the D-loop of tRNA on the catalytic, anticodon-binding, and Add-1 domains of ArgRS also requires some flexibility of the tRNA molecule, provided by G:U base pairs, to achieve the productive conformation of the active site of the enzyme by induced fit.     

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA^Leu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA^Leu (CAG)—a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.     

Transfer RNA-Peroxidase Interaction: INHIBITION OF INDOLE-3-ACETIC ACID OXIDATION

Transfer RNA from wheat germ, yeast, and Escherichia coli inhibited the indoleacetic acid (IAA)-induced spectral change in horseradish peroxidase (EC 1.11.1.7) and the decarboxylation of IAA. The inhibition was limited to a delay after which the increase in A₄₂₇ and the decarboxylation of IAA resumed at the same rate as in the control; the duration of the inhibition was dependent on, but not proportional to, the concentration of tRNA. Alkaline hydrolysis destroyed the inhibitory activity of tRNA. The inhibition was completely abolished when the tRNA was added 30 seconds after IAA. Thus, the tRNA appears not to react with the enzyme intermediates formed during the reaction with IAA. The inhibition by tRNA was rapidly reversed by H₂O₂ or additional IAA, but not by 2,4-dichlorophenol. Results suggest that the tRNA interferes with the initial reaction between IAA and the heme moiety of free peroxidase, thus preventing the formation of highly active enzyme intermediates essential for IAA degradation.     

tRNAGlu Increases the Affinity of Glutamyl-tRNA Synthetase for Its Inhibitor Glutamyl-Sulfamoyl-Adenosine,an Analogue of the Aminoacylation Reaction Intermediate Glutamyl-AMP: Mechanistic and Evolutionary Implications

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNA^Glu or of the non-cognate tRNA^Phe is indicated by the negative values of –TΔS_b, and by the negative value of ΔC_p. On the other hand, the large negative enthalpy is the dominant contribution to ΔG_b in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNA^Phe, but the dissociation constant K _d is decreased 50-fold in the presence of tRNA^Glu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNA^Glu in the Glu-AMS•GluRS•tRNA^Glu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNA^Glu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.     

Two forms of methionyl-transfer RNA synthetase from Mycobacterium smegmatis

Two methionyl-transfer RNA synthetases (A and B forms) have been isolated from $\text{Mycobacterium smegmatis}$. The homogeneous preparations of the enzymes showed 1500 fold increase in specific activity in aminoacylation of methionine specific tRNA. The A and B forms differed in their specificity of aminoacylation of tRNA_m^Met and tRNA_f^Met; enzyme B exhibited much higher specificity for tRNA_f^Met. The molecular activities of A and B enzymes for aminoacid and tRNA were identical. The turnover number for aminoacid was 27 fold greater than that for tRNA, while the Km values for tRNA were lower by a factor of 10⁶ as compared to the aminoacid. Both the enzymes catalysed ATP-pyrophosphate exchange reaction to the same extent.