Active site mapping of yeast aspartyl-tRNA synthetase by in vivo selection of enzyme mutations lethal for cell growth.

The active site of yeast aspartyl-tRNA synthetase has been characterised by structural and functional approaches. However, residues or structural elements that indirectly contribute to the active site organisation have still to be described. They have not been assessed by simple analysis of structural data or site-directed mutagenesis analysis, since rational targetting has proven difficult. Here, we attempt to locate these functional features by using a genetic selection method to screen a randomly mutated yeast AspRS library for mutations lethal for cell growth. This approach is an efficient method to map the active site residues, since of the 23 different mutations isolated, 13 are in direct contact with the substrates. Most of the mutations are located in a 15 A radius sphere around the ATP molecule, where they affect the very conserved residues of the class-defining motifs. The results also showed the importance of the dimer interface for the enzyme activity: a single mutation of the invariant proline residue of motif 1 led to a structural defect inactivating the enzyme. From in vivo complementation studies it appeared that the enzyme activity can be recovered by reconstitution of an intact interface through the formation of heterodimers. We also show that a single mutation affecting an interaction with G34 of the tRNA can inactivate the enzyme by inducing a relaxation of the tRNA recognition specificity. Finally, several mutants whose functional importance could not be assessed from the structural data were selected, demonstrating the importance of this type of approach in the context of a structure-function relationship study.     

A single catalytically active subunit in the multimeric Sulfolobus shibatae CCA-adding enzyme can carry out all three steps of CCA addition

The CCA-adding enzyme ATP(CTP):tRNA nucleotidyltransferase builds and repairs the 3'-terminal CCA sequence of tRNA. Although this unusual RNA polymerase has no nucleic acid template, it can construct the CCA sequence one nucleotide at a time using CTP and ATP as substrates. We found previously that tRNA does not translocate along the enzyme during CCA addition (Yue, D., Weiner, A. M., and Maizels, N. (1998) J. Biol. Chem. 273, 29693-29700) and that a single nucleotidyltransferase motif adds all three nucleotides (Shi, P.-Y., Maizels, N., and Weiner, A. M. (1998) EMBO J. 17, 3197-3206). Intriguingly, the CCA-adding enzyme from the archaeon Sulfolobus shibatae is a homodimer that forms a tetramer upon binding two tRNAs. We therefore asked whether the active form of the S. shibatae enzyme might have two quasi-equivalent active sites, one adding CTP and the other ATP. Using an intersubunit complementation approach, we demonstrate that the dimer is active and that a single catalytically active subunit can carry out all three steps of CCA addition. We also locate one UV light-induced tRNA cross-link on the enzyme structure and provide evidence suggesting the location of another. Our data rule out shuttling models in which the 3'-end of the tRNA shuttles from one quasi-equivalent active site to another, demonstrate that tRNA-induced tetramerization is not required for CCA addition, and support a role for the tail domain of the enzyme in tRNA binding.     

Fold recognition,homology modeling,docking simulations,kinetics analysis and mutagenesis of ATP/CTP:tRNA nucleotidyltransferase from Methanococcus jannaschii

ATP/CTP:tRNA nucleotidyltransferases (NTases) and poly(A) polymerases (PAPs) belong to the same superfamily and their catalytic domains are remotely related. Based on the results of fold-recognition analysis and comparison of secondary structure patterns, we predicted that these two NTase families share three domains, corresponding to "palm," "fingers," and "fingernails" in the PAP crystal structure. A homology model of tRNA NTase from Methanococcus jannaschii was constructed. Energy minimization calculations of enzyme-nucleotide complexes and computer-aided docking of nucleotides onto the enzyme's surface were carried out to explore possible ATP and CTP binding sites. Theoretical models were used to guide experimental analysis. Recombinant His-tagged enzyme was expressed in Escherichia coli, and kinetic properties were characterized. The apparent K(M) for CTP was determined to be 38 microM, and the apparent K(M) for ATP was 21 microM. Three mutations of basic amino acids to alanine were created in a highly conserved region predicted to be in the vicinity of the nucleotide binding site. A deletion was also constructed to remove the C-terminal structural domain defined by the model; it retained about 1% of wild type enzymatic activity using CTP as co-substrate, confirming that detectable catalytic activity is exhibited by the N-terminal domain, as defined by the model. Our results suggest a mechanism of differential ATP and CTP binding, which explains how the tRNA NTase, having only one catalytic site, utilizes different nucleotide triphosphates depending on the nature of the tRNA substrate.     

Archaeal CCA-adding enzymes: central role of a highly conserved beta-turn motif in RNA polymerization without translocation

The CCA-adding enzyme (tRNA nucleotidyltransferase) builds and repairs the 3' end of tRNA. A single active site adds both CTP and ATP, but the enzyme has no nucleic acid template, and tRNA does not translocate or rotate during C75 and A76 addition. We modeled the structure of the class I archaeal Sulfolobus shibatae CCA-adding enzyme on eukaryotic poly(A) polymerase and mutated residues in the vicinity of the active site. We found mutations that specifically affected C74, C75, or A76 addition, as well as mutations that progressively impaired addition of CCA. Many of these mutations clustered in an evolutionarily versatile beta-turn located between strands 3 and 4 of the nucleotidyltransferase domain. Our mutational analysis confirms and extends recent crystallographic studies of the highly homologous Archaeoglobus fulgidus enzyme. We suggest that the unusual phenotypes of the beta-turn mutants reflect the consecutive conformations assumed by the beta-turn as it presents the discriminator base N73, then C74, and finally C75 to the active site without translocation or rotation of the tRNA acceptor stem. We also suggest that beta-turn mutants can affect nucleotide selection because the growing 3' end of tRNA must be properly positioned to serve as part of the ribonucleoprotein template that selects the incoming nucleotide.     

Structural and functional analyses of the human-type corrinoid adenosyltransferase (PduO) from Lactobacillus reuteri

ATP:Co(I)rrinoid adenosyltransferase (ACA) catalyzes the conversion of cobalamin to coenzyme B12, an essential cofactor in animal metabolism. Several mutations of conserved residues in the active site of human ACA have been identified in humans with methylmalonic aciduria. However, the catalytic role of these residues remains unclear. To better understand the function of these residues and to determine how the enzyme promotes catalysis, several variants of a human-type ACA from the lactic acid bacterium Lactobacillus reuteri (LrPduO) were kinetically and structurally characterized. Kinetic analyses of a series of alternate nucleotides were also performed. Substrate inhibition was observed at subsaturating concentrations of ATP, consistent with an ordered binding scheme where ATP is bound first by the enzyme. Modification or elimination of an active site, inter-subunit salt bridge resulted in a reduced "on" rate for ATP binding, with a less significant disruption in the rate of subsequent catalytic steps. Kinetic and structural data demonstrate that residue Arg132 is not involved in orienting ATP in the active site but, rather, it stabilizes the altered substrate in the transition state. Two functional groups of ATP explain the reduced ability of the enzyme to use alternate nucleotides: the amino group at the C-6 position of ATP contributes approximately 6 kcal/mol of free energy to ground state binding, and the nitrogen at the N-7 position assists in coordinating the magnesium ion in the active site. This study provides new insight into the role of substrate binding determinants and active site residues in the reaction catalyzed by a human-type ACA.     

Conformational movements and cooperativity upon amino acid,ATP and tRNA binding in threonyl-tRNA synthetase

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8A at some C(alpha) positions, of a strand-loop-strand region of the core beta-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.     

Yeast phenylalanyl-tRNA synthetase. Affinity and photoaffinity labelling of the stereospecific binding sites.

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The RNAPhe was cross-linked to the enzyme by non-specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (S4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate-oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation. The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA. The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the beta subunit (Mr 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to eigher type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.     

Dual mechanisms for glucose 6-phosphate inhibition of human brain hexokinase.

Brain hexokinase (HKI) is inhibited potently by its product glucose 6-phosphate (G6P); however, the mechanism of inhibition is unsettled. Two hypotheses have been proposed to account for product inhibition of HKI. In one, G6P binds to the active site (the C-terminal half of HKI) and competes directly with ATP, whereas in the alternative suggestion the inhibitor binds to an allosteric site (the N-terminal half of HKI), which indirectly displaces ATP from the active site. Single mutations within G6P binding pockets, as defined by crystal structures, at either the N- or C-terminal half of HKI have no significant effect on G6P inhibition. On the other hand, the corresponding mutations eliminate product inhibition in a truncated form of HKI, consisting only of the C-terminal half of the enzyme. Only through combined mutations at the active and allosteric sites, using residues for which single mutations had little effect, was product inhibition eliminated in HKI. Evidently, potent inhibition of HKI by G6P can occur from both active and allosteric binding sites. Furthermore, kinetic data reported here, in conjunction with published equilibrium binding data, are consistent with inhibitory sites of comparable affinity linked by a mechanism of negative cooperativity.     

Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.

Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.     

Structural plasticity and enzyme action: crystal structures of mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacterium tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme from Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body of the molecule and a polypeptide stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule.     

Structure of the yeast isoleucyl-tRNA synthetase gene (ILS1). DNA-sequence, amino-acid sequence of proteolytic peptides of the enzyme and comparison of the structure to those of other known aminoacyl-tRNA synthetases

The ILS1 gene encoding for cytoplasmic isoleucyl-tRNA synthetase from Saccharomyces cerevisiae was subcloned from a 5.4-kb insert of the shuttle vector YEp13 to M13mp8 and M13mp9. Nucleotide sequence analysis of a 4.3-kb BamHI-HpaI fragment revealed a single open reading frame from which we deduced the amino-acid sequence of the enzyme. Independently obtained amino-acid sequence information from ten tryptic peptides of the purified enzyme confirmed the gene-derived structure. The enzyme is comprised of 1073 amino-acids consistent with earlier determinations of its molecular mass. The codon usage of ILS1 is typical of abundant yeast proteins. A significant homology to E. coli isoleucyl- and valyl-tRNA synthetases as well as to yeast valyl-tRNA synthetase was detected. The characteristic amino-acid residues of the aminoacyl-adenylate site and of the potential binding site of the 3'-end of tRNA found in other synthetases are present in the structure.     

Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase.

The crystal structure of a fully biologically active monomeric form of Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990) J. Mol. Biol. 216, 411-424), revealing details of the active site of the enzyme, including the location of amino acid residues potentially involved in substrate binding. In the present paper, the role of 3 active site residues in interaction with methionine, ATP, and tRNA(fMet) and in catalysis of methionyl-adenylate has been explored using site-directed mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala produced a 14-fold increase in the Km for ATP with only a small (2-3-fold) change in the other kinetic parameters, indicating that the major role of this residue is in formation of the initial complex with ATP and/or in stabilization of the methionyl-adenylate reaction intermediate. Mutation of the adjacent residue Tyr358 to Ala had no effect on the Km values for methionine or ATP but produced nearly a 2000-fold decrease in the rate of ATP-PPi exchange. This mutation also dramatically reduced the rate of pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of pyrophosphate without increasing the Km for PPi. None of the mutations affected the Km for tRNAfMet in the aminoacylation reaction. The results suggest that Tyr358 may enhance the rate of methionyl-adenylate formation by binding to the alpha-phosphate of ATP in the transition state. Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction requires a significant change in the conformation of this region of the active site compared to the structure found in the MetRS.ATP complex. Such a shift is consistent with an induced-fit mechanism for methionine activation. Primary sequence comparisons of methionine-specific enzymes from yeast and bacterial sources reveals that Tyr358 is conserved in all of the known MetRS sequences.     

Role of Lys-32 residues in R67 dihydrofolate reductase probed by asymmetric mutations

R67 dihydrofolate reductase (R67 DHFR) is a novel protein encoded by an R-plasmid that confers resistance to the antibiotic, trimethoprim. This homotetrameric enzyme possesses 222 symmetry, which imposes numerous constraints on the single active site pore, including a "one-site-fits-both" strategy for binding its ligands, dihydrofolate (DHF) and NADPH. Previous studies uncovered salt effects on binding and catalysis (Hicks, S. N., Smiley, R. D., Hamilton, J. B., and Howell, E. E. (2003) Biochemistry 42, 10569-10578), however the one or more residues that participate in ionic contacts with the negatively charged tail of DHF as well as the phosphate groups in NADPH were not identified. Several studies predict that Lys-32 residues were involved, however mutations at this residue destabilize the R67 DHFR homotetramer. To study the role of Lys-32 in binding and catalysis, asymmetric K32M mutations have been utilized. To create asymmetry, individual mutations were added to a tandem array of four in-frame gene copies. These studies show one K32M mutation is tolerated quite well, whereas addition of two mutations has variable effects. Two double mutants, K32M:1+2 and K32M: 1+4, which place the mutations on opposite sides of the pore, reduce kcat. However a third double mutant, K32M: 1+3, that places two mutations on the same half pore, enhances kcat 4- to 5-fold compared with the parent enzyme, albeit at the expense of weaker binding of ligands. Because the kcat/Km values for this double mutant series are similar, these mutations appear to have uncovered some degree of non-productive binding. This non-productive binding mode likely arises from formation of an ionic interaction that must be broken to allow access to the transition state. The K32M:1+3 mutant data suggest this interaction is an ionic interaction between Lys-32 and the charged tail of dihydrofolate. This unusual catalytic scenario arises from the 222 symmetry imposed on the single active site pore.     

Detailed characterization of the cooperative mechanism of Ca(2+) binding and catalytic activation in the Ca(2+) transport (SERCA) ATPase

Expression of heterologous SERCA1a ATPase in Cos-1 cells was optimized to yield levels that account for 10-15% of the microsomal protein, as revealed by protein staining on electrophoretic gels. This high level of expression significantly improved our characterization of mutants, including direct measurements of Ca(2+) binding by the ATPase in the absence of ATP, and measurements of various enzyme functions in the presence of ATP or P(i). Mutational analysis distinguished two groups of amino acids within the transmembrane domain: The first group includes Glu771 (M5), Thr799 (M6), Asp800 (M6), and Glu908 (M8), whose individual mutations totally inhibit binding of the two Ca(2+) required for activation of one ATPase molecule. The second group includes Glu309 (M4) and Asn796 (M6), whose individual or combined mutations inhibit binding of only one and the same Ca(2+). The effects of mutations of these amino acids were interpreted in the light of recent information on the ATPase high-resolution structure, explaining the mechanism of Ca(2+) binding and catalytic activation in terms of two cooperative sites. The Glu771, Thr799, and Asp800 side chains contribute prominently to site 1, together with less prominent contributions by Asn768 and Glu908. The Glu309, Asn796, and Asp800 side chains, as well as the Ala305 (and possibly Val304 and Ile307) carbonyl oxygen, contribute to site 2. Sequential binding begins with Ca(2+) occupancy of site 1, followed by transition to a conformation (E') sensitive to Ca(2+) inhibition of enzyme phosphorylation by P(i), but still unable to utilize ATP. The E' conformation accepts the second Ca(2+) on site 2, producing then a conformation (E' ') which is able to utilize ATP. Mutations of residues (Asp813 and Asp818) in the M6/M7 loop reduce Ca(2+) affinity and catalytic turnover, suggesting a strong influence of this loop on the correct positioning of the M6 helix. Mutation of Asp351 (at the catalytic site within the cytosolic domain) produces total inhibition of ATP utilization and enzyme phosphorylation by P(i), without a significant effect on Ca(2+) binding.     

The tRNA recognition mechanism of the minimalist SPOUT methyltransferase,TrmL

Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Both the catalytic and tRNA recognition mechanisms of this enzyme remain elusive. By using tRNAs purified from an Escherichia coli strain with the TrmL gene deleted, we found that TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to and isoacceptors without the involvement of other tRNA-binding proteins. We have solved the crystal structures of TrmL in apo form and in complex with S-adenosyl-homocysteine and identified the cofactor binding site and a possible active site. Methyltransferase activity and tRNA-binding affinity of TrmL mutants were measured to identify residues important for tRNA binding of TrmL. Our results suggest that TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition.     

RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase

In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.     

Mutation and evolution of the magnesium-binding site of a class II aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases contain one or three Mg(2+) ions in their catalytic sites. In addition to their role in ATP binding, these ions are presumed to play a role in catalysis by increasing the electropositivity of the alpha-phosphate and stabilizing the pentavalent transition state. In the class II aaRS, two highly conserved carboxylate residues have been shown to participate with Mg(2+) ions in binding and coordination. It is shown here that these carboxylate residues are absolutely required for the activity of Saccharomyces cerevisiae aspartyl-tRNA synthetase. Mutants of these residues exhibit pleiotropic effects on the kinetic parameters suggesting an effect at an early stage of the aminoacylation reaction, such as the binding of ATP, Mg(2+), aspartic acid, or the amino acid activation. Despite genetic selections in an APS-knockout yeast strain, we were unable to select a single active mutant of these carboxylate residues. Nevertheless, we isolated an intragenic suppressor from a combinatorial library. The active mutant showed a second substitution close to the first one, and exhibited a significant increase of the tRNA aminoacylation rate. Structural analysis suggests that the acceptor stem of the tRNA might be repositioned to give a more productive enzyme:tRNA complex. Thus, the initial defect of the activation reaction was compensated by a significant increase of the aminoacylation rate that led to cellular complementation.     

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.