tRNA Aminoacylation Reaction Research Articles

Extracellular tyrosyl‐tRNA synthetase cleaved by plasma proteinases and stored in platelet α‐granules: Potential role in monocyte activation

BackgroundTyrosyl‐tRNA synthetase (YRS) belongs to the family of enzymes that catalyzes the tRNA aminoacylation reaction for protein synthesis, and it has been recently shown to exert noncanonical functions. Although database results indicate extremely low levels of YRS mRNA in platelets, YRS protein is abundantly present. The source of YRS in platelets, as well as the physiological role of platelet‐stored YRS, remains largely unknown. ObjectivesTo clarify how YRS accumulates in platelets and determine the potential role of platelet‐stored YRS. MethodsRecombinant YRS proteins with epitope tags were prepared and tested in vitro for proteolytic cleavage in human plasma. Fluorescent‐labeled YRS was examined for uptake by platelets, as demonstrated by western blotting and confocal microscopy analysis. Using RAW‐Dual reporter cells, Toll‐like receptor and type I interferon activation pathways were analyzed after treatment with YRS. ResultsFull‐length YRS was cleaved by both elastase and matrix metalloproteinases in the plasma. The cleaved, N‐terminal YRS fragment corresponds to the endogenous YRS detected in platelet lysate by western blotting. Both full‐length and cleaved forms of YRS were taken up by platelets in vitro and stored in the α‐granules. The N‐terminal YRS fragment generated by proteolytic cleavage had monocyte activation comparable to that of the constitutive‐active mutant YRS (YRSY341A) previously reported. ConclusionPlatelets take up both full‐length YRS and the active form of cleaved YRS fragment from the plasma. The cleaved, N‐terminal YRS fragment stored in α‐granules may have potential to activate monocytes.

Expanding a tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. In general, aminoacyl-tRNA synthetase assays require stoichiometric amounts of tRNA, which limits their sensitivity while increasing their cost. This requirement for stoichiometric amounts of tRNA can be alleviated if the aminoacyl-tRNA product is cleaved following the tRNA aminoacylation reaction, regenerating the free tRNA substrate. This data article is related to the research article entitled “A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate” in which this approach is used to develop a continuous spectrophotometric assay for tyrosyl-tRNA synthetase [1]. Here we present enzymes that can be used to cleave the aminoacyl-tRNA product for at least 16 of the 20 naturally occurring amino acids. These enzymes can be used to extend the tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases.

Comparison of the Intrinsic Dynamics of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617-650) [1]. AARSs are grouped into two broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617-650) [1]. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia-c and IIa-c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes--(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.

The mechanistic and evolutionary aspects of the 2′- and 3′-OH paradigm in biosynthetic machinery

BackgroundThe translation machinery underlies a multitude of biological processes within the cell. The design and implementation of the modern translation apparatus on even the simplest course of action is extremely complex, and involves different RNA and protein factors. According to the “RNA world” idea, the critical link in the translation machinery may be assigned to an adaptor tRNA molecule. Its exceptional functional and structural characteristics are of primary importance in understanding the evolutionary relationships among all these macromolecular components.Presentation of the hypothesisThe 2′-3′ hydroxyls of the tRNA A76 constitute chemical groups of critical functional importance, as they are implicated in almost all phases of protein biosynthesis. They contribute to: a) each step of the tRNA aminoacylation reaction catalyzed by aminoacyl-tRNA synthetases (aaRSs); b) the isomerase activity of EF-Tu, involving a mixture of the 2′(3′)- aminoacyl tRNA isomers as substrates, thereby producing the required combination of amino acid and tRNA; and c) peptide bond formation at the peptidyl transferase center (PTC) of the ribosome. We hypothesize that specific functions assigned to the 2′-3′ hydroxyls during peptide bond formation co-evolved, together with two modes of attack on the aminoacyl-adenylate carbonyl typical for two classes of aaRSs, and alongside the isomerase activity of EF-Tu. Protein components of the translational apparatus are universally recognized as being of ancient origin, possibly replacing RNA-based enzymes that may have existed before the last universal common ancestor (LUCA). We believe that a remnant of these processes is still imprinted on the organization of modern-day translation.Testing and implications of the hypothesisEarlier publications indicate that it is possible to select ribozymes capable of attaching the aa-AMP moiety to RNA molecules. The scenario described herein would gain general acceptance, if a ribozyme able to activate the amino acid and transfer it onto the terminal ribose of the tRNA, would be found in any life form, or generated in vitro. Interestingly, recent studies have demonstrated the plausibility of using metals, likely abandoned under primordial conditions, as biomimetic catalysts of the aminoacylation reaction.ReviewersThis article was reviewed by Henri Grosjean, Manuel Santos and Eugene Koonin. For complete reviews, go to the Reviewers’ reports section.

Catalysis of tRNA Aminoacylation: Single Turnover to Steady-State Kinetics of tRNA Synthetases

Aminoacyl-tRNA synthetases (aaRS) catalyze the bimolecular association reaction between amino acid and tRNA by specifically and unerringly choosing the cognate amino acid and tRNA. There are two classes of such synthetases that perform tRNA-aminoacylation reaction. Interestingly, these two classes of aminoacyl-tRNA synthetases differ not only in their structures but they also exhibit remarkably distinct kinetics under pre-steady-state condition. The class I synthetases show initial burst of product formation followed by a slower steady-state rate. This has been argued to represent the influence of slow product release. In contrast, there is no burst in the case of class II enzymes. The tight binding of product with enzyme for class I enzymes is correlated with the enhancement of rate in presence of elongation factor EF-TU. In spite of extensive experimental studies, there is no detailed theoretical analysis that can provide a quantitative understanding of this important problem. In this article, we present a theoretical investigation of enzyme kinetics for both classes of aminoacyl-tRNA synthetases. We present an augmented kinetic scheme and then employ the methods of time-dependent probability statistics to obtain expressions for the first passage time distribution that gives both the time-dependent and the steady-state rates. The present study quantitatively explains all the above experimental observations. We propose an alternative path way in the case of class II enzymes showing the tRNA-dependent amino acid activation and the discrepancy between the single-turnover and steady-state rate.

Thermodynamic Analysis Reveals a Temperature-dependent Change in the Catalytic Mechanism of Bacillus stearothermophilus Tyrosyl-tRNA Synthetase

Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase can be divided into two steps. In the first step, tyrosine is activated by ATP to form the tyrosyl-adenylate intermediate. In the second step, the tyrosyl moiety is transferred to the 3' end of tRNA. To investigate the roles that enthalpic and entropic contributions play in catalysis by Bacillus stearothermophilus tyrosyl-tRNA synthetase (TyrRS), the temperature dependence for the activation of tyrosine and subsequent transfer to tRNA(Tyr) has been determined using single turnover kinetic methods. A van't Hoff plot for binding of ATP to the TyrRS.Tyr complex reveals three distinct regions. Particularly striking is the change occurring at 25 degrees C, where the values of DeltaH(0) and DeltaS(0) go from -144 kJ/mol and -438 J/mol K below 25 degrees C to +137.9 kJ/mol and +507 J/mol K above 25 degrees C. Nonlinear Eyring and van't Hoff plots are also observed for formation of the TyrRS.[Tyr-ATP](double dagger) and TyrRS.Tyr-AMP complexes. Comparing the van't Hoff plots for the binding of ATP to tyrosyl-tRNA synthetase in the absence and presence of saturating tyrosine concentrations indicates that the temperature-dependent changes in DeltaH(0) and DeltaS(0) for the binding of ATP only occur when tyrosine is bound to the enzyme. Previous investigations revealed a similar synergistic interaction between the tyrosine and ATP substrates when the "KMSKS" signature sequence is deleted or replaced by a nonfunctional sequence. We propose that the temperature-dependent changes in DeltaH(0) and DeltaS(0) are because of the KMSKS signature sequence being conformationally constrained and unable to disrupt this synergistic interaction below 25 degrees C.

Solid-Phase Combinatorial Synthesis of a Lysyl-tRNA Synthetase (LysRS) Inhibitory Library

The solid-phase combinatorial synthesis of a new library with potential inhibitory activity against the cytoplasmic lysyl-tRNA synthetase (LysRS) isoform of Trypanosoma brucei is described. The library has been specifically designed to mimic the lysyl adenylate complex. The design was carried out by dividing the complex into four modular parts. Proline derivatives (cis-gamma-amino-L-proline or trans-gamma-hydroxy-L-proline) were chosen as central scaffolds. After primary screening, three compounds of the library caused in vitro inhibition of the tRNA aminoacylation reaction in the low micromolar range.

Enzymatic tRNA Acylation by Acid and α-Hydroxy Acid Analogues of Amino Acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.

The tRNA-Interacting Factor p43 Associates with Mammalian Arginyl-tRNA Synthetase but Does Not Modify Its tRNA Aminoacylation Properties

Arginyl-tRNA synthetase (ArgRS) is one of the nine synthetase components of a multienzyme complex containing three auxiliary proteins as well. We previously established that the N-terminal moiety of the auxiliary protein p43 associates with the N-terminal, eukaryotic-specific polypeptide extension of ArgRS. Because p43 is homologous to Arc1p, a yeast general RNA-binding protein that associates with MetRS and GluRS and plays the role of tRNA-binding cofactor in the aminoacylation reaction, we analyzed the functional significance of p43-ArgRS association. We had previously showed that full-length ArgRS, corresponding to the ArgRS species associated within the multisynthetase complex, and ArgRS with a deletion of 73 N-terminal amino acid residues, corresponding to a free species of ArgRS, both produced in yeast, have similar catalytic parameters (Lazard, M., Kerjan, P., Agou, F., and Mirande, M. (2000) J. Mol. Biol. 302, 991-1004). However, a recent study had suggested that association of p43 to ArgRS reduces the apparent K(M) of ArgRS to tRNA (Park, S. G., Jung, K. H., Lee, J. S., Jo, Y. J., Motegi, H., Kim, S., and Shiba, K. (1999) J. Biol. Chem. 274, 16673-16676). In this study, we analyzed in detail, by gel retardation assays and enzyme kinetics, the putative role of p43 as a tRNA-binding cofactor of ArgRS. The association of p43 with ArgRS neither strengthened tRNA-binding nor changed kinetic parameters in the amino acid activation or in the tRNA aminoacylation reaction. Furthermore, selective removal of the C-terminal RNA-binding domain of p43 from the multisynthetase complex did not affect kinetic parameters for ArgRS. Therefore, p43 has a dual function. It promotes association of ArgRS to the complex via its N-terminal domain, but its C-terminal RNA-binding domain may act as a tRNA-interacting factor for an as yet unidentified component of the complex.

Using a Solid-Phase Ribozyme Aminoacylation System to Reprogram the Genetic Code

Here, we report a simple and economical tRNA aminoacylation system based upon a resin-immobilized ribozyme, referred to as Flexiresin. This catalytic system features a broad spectrum of activities toward various phenylalanine (Phe) analogs and suppressor tRNAs. Most importantly, it allows users to perform the tRNA aminoacylation reaction and isolate the aminoacylated tRNAs in a few hours. We coupled the Flexiresin system with a high-performance cell-free translation system and demonstrated protein mutagenesis with seven different Phe analogs in parallel. Thus, the technology developed herein provides a new tool that significantly simplifies the procedures for the synthesis of aminoacyl-tRNAs charged with nonnatural amino acids, which makes the nonnatural amino acid mutagenesis of proteins more user accessible .

Transition State Stabilization by the N-terminal Anticodon-binding Domain of Lysyl-tRNA Synthetase

Lysyl-tRNA synthetase from Bacillus stearothermophilus (B.s. LysRS) (EC ) catalyzes aminoacylation of tRNA(Lys) with l-lysine, in which l-lysine was first activated with ATP to yield an enzyme (lysyladenylate complex), and then the lysine molecule was transferred from the complex to tRNA(Lys). B.s. LysRS is a homodimeric enzyme with a subunit that consists of two domains, an N-terminal tRNA anticodon-binding domain (TAB-ND: Ser(1)-Pro(144)) and a C-terminal Class II-specific catalytic domain (CAT-CD: Lys(151)-Lys(493)). CAT-CD alone retained catalytic activity, although at a low level; TAB-ND alone showed no activity. Size exclusion chromatography revealed that CAT-CD exists as a dimer, whereas TAB-ND was a monomer. The formation of a complex consisting of these domains was detected with the guidance of surface plasmon resonance. In accordance with this, the addition of TAB-ND to CAT-CD significantly enhanced both the l-lysine activation and the tRNA aminoacylation reactions. Kinetic analysis showed that deletion of TAB-ND resulted in a significant destabilization of the transition state of CAT-CD in the l-lysine activation reaction but had little effect on the ground state of substrate binding. A significant role of a cross-subunit interaction in the enzyme between TAB-ND and CAT-CD was proposed for the stabilization of the transition state in the l-lysine activation reaction.

Correlating amino acid conservation with function in tyrosyl-tRNA synthetase

Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNA Tyr aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA Tyr. Previous investigations indicate that the class I conserved KMSKS motif is involved in only the first step of the reaction (i.e. tyrosine activation). Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step. In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction. These results imply that stabilization of the transition state for the first step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 3′ end of tRNA. We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP.

The tRNA-dependent activation of arginine by arginyl-tRNA synthetase requires inter-domain communication

The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and purified. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP i exchange and tRNA aminoacylation reactions. This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the α-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site.

Function of the extra 5'-phosphate carried by histidine tRNA.

Among elongator tRNAs, tRNA specific for histidine has the peculiarity to possess one extra nucleotide at position -1. This nucleotide is believed to be responsible for recognition by histidyl-tRNA synthetase. Here, we show that, in fact, it is the phosphate 5' to the extra nucleotide which mainly supports the efficiency of the tRNA aminoacylation reaction catalyzed by Escherichia coli histidyl-tRNA synthetase. In the case of the reaction of E. coli peptidyl-tRNA hydrolase, this atypical phosphate is dispensable. Instead, peptidyl-tRNA hydrolase recognizes the phosphate of the phosphodiester bond between residues -1 and +1 of tRNA(His). Recognition of the +1 phosphate of tRNA(His) by peptidyl-tRNA hydrolase resembles, therefore, that of the 5'-terminal phosphate of other elongator tRNAs.

Misacylation of tRNALys with noncognate amino acids by lysyl-tRNA synthetase.

Lysyl-tRNA synthetase (LysRS), a class II enzyme whose major function is to provide Lys-tRNALys for protein synthesis, also catalyzes aminoacylation of tRNALys with arginine, threonine, methionine, leucine, alanine, serine, and cysteine. The limited selectivity in the tRNA aminoacylation reaction appears to be due to inefficient editing of some amino acids (Met, Leu, Cys, Ala, Thr) by a pre-transfer mechanism or the absence of editing of other amino acids (Arg and Ser). Purified Arg-tRNALys, Thr-tRNALys, and Met-tRNALys were essentially not deacylated by LysRS, indicating that the enzyme does not possess a post-transfer editing mechanism. However, LysRS possesses an efficient pre-transfer editing mechanism which prevents misacylation of tRNALys with ornithine. A novel feature of this editing reaction is that ornithine lactam is formed by the facile cyclization of ornithyl adenylate.

A peculiarity of the reaction of tRNA aminoacylation catalyzed by phenylalanyl-tRNA synthetase from the extreme thermophile Thermus thermophilus

It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation reaction catalyzed by Thermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two bound phenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quite low: the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. The thermophilic synthetase can aminoacylate twice not only T. thermophilus tRNA Phe but also Escherichia coli tRNA Phe and E. coli tRNA Phe transcript, indicating that the presence of modified nucleotides is not necessary for tRNA Phe overcharging. Bisphenylalanyl-tRNA is stable in acidic solution, but it decomposes in alkaline medium yielding finally tRNA and free phenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate as from monophenylalanyl-tRNA. In the presence of the enzyme the rate of bisphenylalanyl-tRNA deacylation increases. Aminoacylated tRNA Phe isolated from T. thermophilus living cells was observed to contain no detectable bisphenylalanyl-tRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed.

Expression of Rat Aspartyl-tRNA Synthetase in Saccharomyces cerevisiae: ROLE OF THE NH2-TERMINAL POLYPEPTIDE EXTENSION ON ENZYME ACTIVITY AND STABILITY

Cytoplasmic aspartyl-tRNA synthetase from mammals is one of the components of a multienzyme complex comprising nine synthetase activities. The presence of an amino-terminal extension composed of about 40 residues is a characteristic of the eukaryotic enzyme. We report here the expression in the yeast Saccharomyces cerevisiae of a native form of rat aspartyl-tRNA synthetase and of two truncated derivatives lacking 20 or 36 amino acid residues from their amino-terminal polypeptide extension. The three recombinant enzyme species were purified to homogeneity. They behave as alpha2 dimers and display catalytic parameters in the tRNA aminoacylation reaction identical to those determined for the native, complex-associated form of aspartyl-tRNA synthetase isolated from rat liver. Because the dimer dissociation constant of rat AspRS is much higher than that of its bacterial and yeast counterparts, we could establish a direct correlation between dissociation of the dimer and inactivation of the enzyme. Our results clearly show that the monomer is devoid of amino acid activation and tRNA aminoacylation activities, indicating that dimerization is essential to confer an active conformation on the catalytic site. The two NH2-terminal truncated derivatives were fully active, but proved to be more unstable than the recombinant native enzyme, suggesting that the polypeptide extension fulfills structural rather than catalytic requirements.

Lysyl-tRNA synthetase from Bacillus stearothermophilus. Purification, and fluorometric and kinetic analysis of the binding of substrates, L-lysine and ATP.

Lysyl-tRNA synthetase [L-lysine:tRNA(Lys)ligase (AMP forming); EC 6.1.1.6] was purified from Bacillus stearothermophilus NCA1503 approximately 1,100-fold to homogeneity in PAGE. The enzyme is a homodimer of M(r) 57,700 x 2. The molar absorption coefficient, epsilon, at 280 nm is 71,600 M-1.cm-1 at pH8.0. Enzyme activity in the tRNA aminoacylation reaction and the ATP-PPi exchange reaction increases up to 50 degrees C at pH 8.0, but is lost completely at 70 degrees C. The pH-optima of the two reactions are 8.3 at 37 degrees C. In the tRNA aminoacylation reaction, the Km values for L-lysine and ATP are 16.4 and 23.2 muM, respectively, and in the ATP-PPi exchange reaction, the Km values for L-lysine and ATP are 23.6 and 65.1 muM, respectively at 37 degrees C, pH 8.0. Interaction of either L-lysine or ATP with the enzyme has been investigated by using as a probe the ligand-induced quenching of protein fluorescence and by equilibrium dialysis. These static analyses, as well as the kinetic analysis of the L-lysine dependent ATP-PPi exchange reaction indicate that the binding mode of L-lysine and ATP to the enzyme is sequential ordered (L-lysine first). The interaction of lysine analogues with the enzyme has also been investigated.

The three cysteine residues of cytoplasmic aspartyl‐tRNA synthetase from Saccharomyces cerevisiae are not essential for its activity

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63,000) each of these containing three cysteines (residues 255, 512 and 519 in the amino acid sequence). Thiol-specific probes were used to label these cysteines and study the resulting effect of the modification on the kinetic parameters of both the ATP/PPi exchange and tRNA aminoacylation reactions. Using the classical techniques of protein chemistry it was shown that none of the three cysteines was labelled with iodoacetic acid, whilst N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoate) reacted with Cys512 and Cys255, respectively. Only the latter modification was accompanied by a decrease in the rates of both enzyme activities whilst the Km values for the various substrates remained unaffected. Site-directed mutagenesis was also used to replace each of the three cysteines by other residues, either individually or simultaneously. For these experiments the enzyme was expressed in Escherichia coli using an expression vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. The resulting substitution in the amino-terminal part of the expressed enzyme had no effect on the kinetic parameters, compared to those of the enzyme purified from S. cerevisiae. Taking into account the consequences of such substitutions, as well as those of chemical modifications on the two reactions catalysed by the enzyme. ATP/PPi exchange and tRNA aminoacylation, it could be concluded that none of these three cysteines plays any essential role in either substrate binding or catalysis.

Static and kinetic studies on binding of a fluorescent analogue of ATP and valyt-tRNA synthetase from Bacillus stearothermophilus

A fluorescent analogue of ATP, 3′-O-antraniloyl-ATP (Ant-ATP), was found to serve as a substrate of valyl-tRNAVal synthetase (l-valine:tRNAVal ligase (AMP-forming), EC 6.1.1.9) from Bacillus stearothermophilus NCA 1503 in the tRNA aminoacylation reaction in place of ATP; Km and kcat at pH 7.5 and 30°C were 440 μM and 0.58 s−1, respectively, whereas those for ATP were 34 μM and 2.9 s−1, respectively. The fluorescence of Ant-ATP (λem = 428 nm) changed significantly on the binding with the enzyme; Kd of the enzyme and Ant-ATP was determined by fluorometric titration to be 290 μM at pH 7.5, 30°C. Fast kinetic studies on the interaction of the enzyme and Ant-ATP were made with a micro-stopped-flow apparatus by using the fluorescence change as probe. The kinetic feature was consistent with a two-step mechanism in which a fast bimolecular process is followed by a unimolecular isomerization process. Kinetic parameters relevant to the mechanism were estimated.