Comparison of the catalytic roles played by the KMSKS motif in the human and Bacillus stearothermophilus trosyl-tRNA synthetases

The Class I aminoacyl-tRNA synthetases are characterized by two signature sequence motifs, "HIGH" and "KMSKS." In Bacillus stearothermophilus tyrosyl-tRNA synthetase, the KMSKS motif (230KFGKT234) has been shown to stabilize the transition state for tyrosine activation through interactions with the pyrophosphate moiety of ATP. In most eukaryotic tyrosyl-tRNA synthetases, the second lysine in the KMSKS motif is replaced by a serine or an alanine residue. Recent kinetic studies indicate that potassium functionally compensates for the absence of the second lysine in the human tyrosyl-tRNA synthetase (222KKSSS226). In this paper, site-directed mutagenesis and pre-steady state kinetics are used to determine the roles that serines 224, 225, and 226 play in catalysis of the tyrosine activation reaction. In addition, the catalytic role played by a downstream lysine conserved in eukaryotic tyrosyl-tRNA synthetases, Lys-231, is investigated. Replacing Ser-224 and Ser-226 with alanine decreases the forward rate constant 7.5- and 60-fold, respectively. In contrast, replacing either Ser-225 or Lys-231 with alanine has no effect on the catalytic activity of the enzyme. These results are consistent with the hypothesis that the KMSSS sequence in human tyrosyl-tRNA synthetase stabilizes the transition state for the tyrosine activation reaction by interacting with the pyrophosphate moiety of ATP. In addition, although they play similar roles in catalysis, the overall contribution of the KMSKS motif to catalysis appears to be significantly less in human tyrosyl-tRNA synthetase than it is in the B. stearothermophilus enzyme.     

Potassium functionally replaces the second lysine of the KMSKS signature sequence in human tyrosyl-tRNA synthetase

Unlike their bacterial homologues, a number of eukaryotic tyrosyl-tRNA synthetases require potassium to catalyze the aminoacylation reaction. In addition, the second lysine in the class I-specific KMSKS signature motif is absent from all known eukaryotic tyrosyl-tRNA synthetase sequences, except those of higher plants. This lysine, which is the most highly conserved residue in the class I aminoacyl-tRNA synthetase family, has been shown to interact with the pyrophosphate moiety of the ATP substrate in the Bacillus stearothermophilus tyrosyl-tRNA synthetase. Equilibrium dialysis and pre-steady-state kinetic analyses were used to determine the role that potassium plays in the tyrosine activation reaction in the human tyrosyl-tRNA synthetase and whether it can be replaced by any of the other alkali metals. Kinetic analyses indicate that potassium interacts with the pyrophosphate moiety of ATP, stabilizing the E.Tyr.ATP and E.[Tyr-ATP] complexes by 2.3 and 4.3 kcal/mol, respectively. Potassium also appears to stabilize the asymmetric conformation of the human tyrosyl-tRNA synthetase dimer by 0.7 kcal/mol. Rubidium is the only other alkali metal that can replace potassium in catalyzing tyrosine activation, although the forward rate constant is half of that observed when potassium is present. The above results are consistent with the hypothesis that potassium functionally replaces the second lysine in the KMSKS signature sequence. Possible implications of these results with respect to the design of antibiotics that target bacterial aminoacyl-tRNA synthetases are discussed.     

Natural variation of tyrosyl-tRNA synthetase and comparison with engineered mutants

We report the cloning and sequence analysis of the gene for the tyrosyl-tRNA synthetase from Bacillus caldotenax and properties of the gene product. The amino acid sequence of the tyrosyl-tRNA synthetase was found to be 99% homologous with the corresponding enzyme from B. stearothermophilus, with only four amino acid differences. Two of these natural variations were found to involve active site residues of the enzyme and correspond to mutations that have been engineered previously in vitro. One, Thr-51----Ala-51, produced a more active enzyme, possessing a higher value of kcat/KM for ATP. Position 51 is a "hot spot" in the tyrosyl-tRNA synthetase, differing in enzymes derived from Escherichia coli, B. stearothermophilus, and B. caldotenax. The other, His-48----Asn-48, is found to be a neutral mutation but is in one of the rare regions that are conserved with other aminoacyl-tRNA synthetases. The equivalence of histidine and asparagine at position 48 extends the homology in this region to more enzymes. These residues, His-Ile-Gly-His, and now His-Ile-Gly-Asn, form part of the binding site for ATP in the transition state of the reaction. Although B. caldotenax is an obligate thermophile with an optimal growth temperature of 80 degrees C, as much as 20 degrees C above the growth optima of strains of Bacillus stearothermophilus, its tyrosyl-tRNA synthetase has an identical thermal stability in vitro to that from B. stearothermophilus.     

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.     

Kinetic and thermodynamic properties of wild-type and engineered mutants of tyrosyl-tRNA synthetase analyzed by pyrophosphate-exchange kinetics.

The first step of the reaction catalyzed by the aminoacyl-tRNA synthetases is the formation of enzyme-bound aminoacyl adenylate. The steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and ATP. A simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identifiable physical processes under a variety of limiting conditions. The free energies of binding to the enzyme of tyrosine, ATP, and the transition state for tyrosyl adenylate formation can be measured in relatively straightforward experiments. The excellent agreement between parameters measured in these experiments and those from earlier pre-steady-state kinetics confirms that the intermediates isolated in the presteady state are kinetically competent. The dissociation constant of ATP from the unligated enzyme, a constant that has previously been experimentally inaccessible, has been measured for wild-type and several mutant enzymes. The changes in enthalpy and entropy of activation on mutation have been measured by a rapid procedure for mutants that have altered contacts with tyrosine and ATP. Those mutants that have large changes of enthalpy and entropy of binding are likely to have structural changes and so warrant further examination by protein crystallography.     

Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 2. Cooperative binding of ATP is limited to the initial turnover of the enzyme

The activation of D-tyrosine by tyrosyl-tRNA synthetase has been investigated using single and multiple turnover kinetic methods. In the presence of saturating concentrations of D-tyrosine, the activation reaction displays sigmoidal kinetics with respect to ATP concentration under single turnover conditions. In contrast, when the kinetics for the activation reaction are monitored using a steady-state (multiple turnover) pyrophosphate exchange assay, Michaelis-Menten kinetics are observed. Previous investigations indicated that activation of l-tyrosine by the K233A variant of Bacillus stearothermophilus tyrosyl-tRNA synthetase displays sigmoidal kinetics similar to those observed for activation of d-tyrosine by the wild-type enzyme. Kinetic analyses indicate that the sigmoidal behavior of the d-tyrosine activation reaction is not enhanced when Lys-233 is replaced by alanine. This supports the hypothesis that the mechanistic basis for the sigmoidal behavior is the same for both d-tyrosine activation by wild-type tyrosyl-tRNA synthetase and activation of l-tyrosine by the K233A variant. The observed sigmoidal behavior presents a paradox, as tyrosyl-tRNA synthetase displays an extreme form of negative cooperativity, known as "half-of-the-sites reactivity," with respect to tyrosine binding and tyrosyl-adenylate formation. We propose that the binding of D-tyrosine weakens the affinity with which ATP binds to the functional subunit in tyrosyl-tRNA synthetase. This allows ATP to bind initially to the nonfunctional subunit, inducing a conformational change in the enzyme that enhances the affinity of the functional subunit for ATP. The observation that sigmoidal kinetics are observed only under single turnover conditions suggests that this conformational change is stable over multiple rounds of catalysis.     

Crystal structure of Staphylococcus aureus tyrosyl-tRNA synthetase in complex with a class of potent and specific inhibitors

SB-219383 and its analogues are a class of potent and specific inhibitors of bacterial tyrosyl-tRNA synthetases. Crystal structures of these inhibitors have been solved in complex with the tyrosyl-tRNA synthetase from Staphylococcus aureus, the bacterium that is largely responsible for hospital-acquired infections. The full-length enzyme yielded crystals that diffracted to 2.8 A resolution, but a truncated version of the enzyme allowed the resolution to be extended to 2.2 A. These inhibitors not only occupy the known substrate binding sites in unique ways, but also reveal a butyl binding pocket. It was reported that the Bacillus stearothermophilus TyrRS T51P mutant has much increased catalytic activity. The S. aureus enzyme happens to have a proline at position 51. Therefore, our structures may contribute to the understanding of the catalytic mechanism and provide the structural basis for designing novel antimicrobial agents.     

Major anticodon-binding region missing from an archaebacterial tRNA synthetase

The small size of the archaebacterial Methanococcus jannaschii tyrosyl-tRNA synthetase may give insights into the historical development of tRNAs and tRNA synthetases. The L-shaped tRNA has two major arms-the acceptor.TpsiC minihelix with the amino acid attachment site and the anticodon-containing arm. The structural organization of the tRNA synthetases parallels that of tRNAs. The more ancient synthetase domain contains the active site and insertions that interact with the minihelix portion of the tRNA. A second, presumably more recent, domain interacts with the anticodon-containing section of tRNA. The small size of the M. jannaschii enzyme is due to the absence of most of the second domain, including a segment thought to bind to the anticodon. Consistent with the absence of an anticodon-binding motif, a mutation of the central base of the anticodon had a relatively small effect on the aminoacylation efficiency of the M. jannaschii enzyme. In contrast, others showed earlier that the same mutation severely reduced charging by a normal-sized bacterial enzyme that has the aforementioned anticodon-binding motif. However, the M. jannaschii enzyme has a peptide insertion into its catalytic domain. This insertion is shared with all other tyrosyl-tRNA synthetases and is needed for a critical minihelix interaction. We show that the M. jannaschii enzyme is active on minihelix substrates over a wide temperature range and has preserved the same peptide-dependent minihelix specificity seen in other tyrosine enzymes. These findings are consistent with the concept that anticodon interactions of tRNA synthetases were later adaptations to the emerging synthetase-tRNA complex that was originally framed around the minihelix.     

Mutants of Escherichia coli with an altered tyrosyl-transfer ribonucleic acid synthetase

We have isolated several mutants defective in the gene for tyrosyl-transfer ribonucleic acid (tRNA) synthetase (tyrS). One of these mutants is described in detail. It was isolated as a tyrosine auxotroph with defects both in the tyrosyl-tRNA synthetase and in the tyrosine biosynthetic enzyme, prephenate dehydrogenase. It also had derepressed levels of the tyrosine-specific 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. The latter finding suggested that a wild-type tyrS gene was required for repression of the tyrosine biosynthetic enzymes. The following results demonstrated that this hypothesis was not correct. (i) When the defective tyrS gene was transferred to another strain, the tyrosine-specific DAHP synthetase in that strain was not derepressed, and (ii) two other mutants with defective tyrosyl-tRNA synthetases had repressed levels of the tyrosine biosynthetic enzymes. The tyrS gene was located near minute 32 on the Escherichia coli chromosome by interrupted mating experiments.     

Cloning and amplified expression of the tyrosyl-tRNA synthetase genes of Bacillus stearothermophilus and Escherichia coli

Two fragments of DNA which carry the genes coding for the tyrosyl-tRNA synthetases of Escherichia coli and Bacillus stearothermophilus have been cloned into the plasmid pBR322 and were selected by complementation of an E. coli temperature-sensitive mutant. Transformation of this strain with either of the recombinant plasmids results in a 100-fold increase in tyrosyl-tRNA synthetase activity measured in vitro and the protein products co-migrate with the corresponding purified enzymes on polyacrylamide gels.     

Protein engineering of homodimeric tyrosyl-tRNA synthetase to produce active heterodimers

Heterodimers of tyrosyl-tRNA synthetase from Bacillus stearothermophilus have been produced by mutagenesis at the subunit interface. Oppositely charged groups have been engineered into the subunits so that they can form a complementary pair. Wild-type tyrosyl-tRNA synthetase is a symmetrical dimer in which the side chains of the 2 Phe-164 residues interact at the subunit interface. Phe-164 was mutated to Asp in tyrosyl-tRNA synthetase and to Lys in a truncated enzyme (des-(321-419)tyrosyl-tRNA synthetase) which lacks the two tRNA-binding sites, but which can catalyze pyrophosphate exchange. The size difference allows subunit association to be studied by gel filtration chromatography. These changes induce reversible dissociation from active dimers into inactive monomers at pH values which favor ionization at position 164. A mixture of the two mutants near neutral pH is apparently fully active in pyrophosphate exchange and consists of a heterodimer of [Asp164]tyrosyl-tRNA synthetase and [Lys164]des-(321-419)tyrosyl-tRNA synthetase. Despite having only one binding site for tRNA, heterodimer has full aminoacylation activity at high concentrations of tyrosine. We have therefore produced a family of dimers that differ in stability near neutral pH. This novel approach using protein engineering allows specific dimerization of subunits of the same size that have different defined mutations, each subunit being tagged by the charge. Such hybrid proteins can be used to study subunit interaction.     

Crystal structure of human mitochondrial tyrosyl-tRNA synthetase reveals common and idiosyncratic features

We report the structure of a strictly mitochondrial human synthetase, namely tyrosyl-tRNA synthetase (mt-TyrRS), in complex with an adenylate analog at 2.2 A resolution. The structure is that of an active enzyme deprived of the C-terminal S4-like domain and resembles eubacterial TyrRSs with a canonical tyrosine-binding pocket and adenylate-binding residues typical of class I synthetases. Two bulges at the enzyme surface, not seen in eubacterial TyrRSs, correspond to conserved sequences in mt-TyrRSs. The synthetase electrostatic surface potential differs from that of other TyrRSs, including the human cytoplasmic homolog and the mitochondrial one from Neurospora crassa. The homodimeric human mt-TyrRS shows an asymmetry propagating from the dimer interface toward the two catalytic sites and extremities of each subunit. Mutagenesis of the catalytic domain reveals functional importance of Ser200 in line with an involvement of A73 rather than N1-N72 in tyrosine identity.     

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.     

Secondary structure of the C-terminal domain of the tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus: a novel type of anticodon binding domain?

The tyrosyl-tRNA synthetase catalyzes the activation of tyrosine and its coupling to the cognate tRNA. The enzyme is made of two domains: an N-terminal catalytic domain and a C-terminal domain that is necessary for tRNA binding and for which it was not possible to determine the structure by X-ray crystallography. We determined the secondary structure of the C-terminal domain of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus by nuclear magnetic resonance methods and found that it is of the alpha+beta type. Its arrangement differs from those of the other anticodon binding domains whose structure is known. We also found that the isolated C-terminal domain behaves as a folded globular protein, and we suggest the presence of a flexible linker between the two domains.     

Crystal structure of yeast acetyl-coenzyme A synthetase in complex with AMP

Acetyl-coenzyme A synthetase (ACS) belongs to the family of AMP-forming enzymes that also includes acyl-CoA synthetases, firefly luciferase, and nonribosomal peptide synthetases. ACS catalyzes the two-step activation of acetate to acetyl-CoA: formation of an acetyl-AMP intermediate from acetate and ATP and the transfer of the acetyl group to CoA. In mammals, the acetyl-CoA product is used for biosynthesis of long chain fatty acids as well as energy production. We have determined the crystal structure of yeast ACS in a binary complex with AMP at 2.3 A resolution. The structure contains a large, N-terminal domain and a small, C-terminal domain. AMP is bound at the interface between the two domains. This structure represents a new conformation for the ACS enzyme, which may be competent for catalyzing the first step of the reaction. A Lys residue that is critical for this step is located in the active site. A rotation of 140 degrees in the small domain is needed for the binding of CoA and the catalysis of the second step. In contrast to the monomeric bacterial enzyme, yeast ACS is a stable trimer.     

Internal thermodynamics of position 51 mutants and natural variants of tyrosyl-tRNA synthetase

Natural variation and evolution impose structural changes on an enzyme that can affect the energetics of catalysis. The energy profile of reaction could, in theory, be altered in three distinct ways: uniform binding changes, differential binding changes, and catalysis of elementary steps. Residue threonine-51 of tyrosyl-tRNA synthetase from Bacillus stearothermophilus is subject to natural variation, being replaced by alanine and proline in the enzymes from Bacillus caldotenax and Escherichia coli, respectively. The consequences of this variation on the energetics of formation of tyrosyl adenylate have been investigated by constructing free energy profiles for wild-type and mutant enzymes constructed by introducing these amino acids into the B. stearothermophilus enzyme. Mutation of Thr-51 to alanine, proline, and cysteine by site-directed mutagenesis improves the stabilization of the transition state in the formation of tyrosyl adenylate. Most marked is the mutation Thr-51----Pro-51 which stabilizes the transition state by 2.2 kcal/mol and accelerates the forward rate 20-fold to a level near that of the enzyme from E. coli. However, the improved transition-state binding is accompanied by an even greater stabilization of tyrosyl adenylate. This reduces the rate of pyrophosphorolysis of tyrosyl adenylate and/or weakens the binding of pyrophosphate in the reverse reaction, shifting the equilibrium between enzyme-bound reactants greatly in favor of the enzyme-intermediate complex. The more stable mutant enzyme-tyrosyl adenylate complexes have lower rates of aminoacylation, suggesting that mutations which stabilize the intermediate slow down the subsequent transfer of tyrosine from tyrosyl adenylate to tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methionyl-tRNA synthetase from Bacillus stearothermophilus: structural and functional identities with the Escherichia coli enzyme.

The metS gene encoding homodimeric methionyl-tRNA synthetase from Bacillus stearothermophilus has been cloned and a 2880 base pair sequence solved. Comparison of the deduced enzyme protomer sequence (Mr 74,355) with that of the E. coli methionyl-tRNA synthetase protomer (Mr 76,124) revealed a relatively low level (32%) of identities, although both enzymes have very similar biochemical properties (Kalogerakos, T., Dessen, P., Fayat, G. and Blanquet, S. (1980) Biochemistry 19, 3712-3723). However, all the sequence patterns whose functional significance have been probed in the case of the E. coli enzyme are found in the thermostable enzyme sequence. In particular, a stretch of 16 amino acids corresponding to the CAU anticodon binding site in the E. coli synthetase structure is highly conserved in the metS sequence. The metS product could be expressed in E. coli and purified. It showed structure-function relationships identical to those of the enzyme extracted from B. stearothermophilus cells. In particular, the patterns of mild proteolysis were the same. Subtilisin converted the native dimer into a fully active monomeric species (62 kDa), while trypsin digestion yielded an inactive form because of an additional cleavage of the 62 kDa polypeptide into two subfragments capable however of remaining firmly associated. The subtilisin cleavage site was mapped on the enzyme polypeptide, and a gene encoding the active monomer was constructed and expressed in E. coli. Finally, trypsin attack was demonstrated to cleave a peptidic bond within the KMSKS sequence common to E. coli and B. stearothermophilus methionyl-tRNA synthetases. This sequence has been shown, in the case of the E. coli enzyme, to have an essential role for the catalysis of methionyl-adenylate formation.     

Fine structure-activity analysis of mutations at position 51 of tyrosyl-tRNA synthetase

Residue Thr-51 at the active site of tyrosyl-tRNA synthetase (Bacillus stearothermophilus) has been replaced with all the smaller amino acids by protein engineering to investigate direct and indirect effects of mutation on substrate binding and catalysis. The gamma-hydroxyl group of Thr-51 was thought to be 0.5 A too far from the ribose ring oxygen of ATP to form a hydrogen bond. Consistent with this, it is found that mutation of Thr-51----Cys-51, which should place the gamma-thiol group within its correct distance for hydrogen bonding, increases the affinity of the enzyme for ATP. Other mutations (Ser-51, Ala-51, and Gly-51) show the contributions to binding of the other atoms in the side chain of Thr-51. A family of enzymes has been produced, TyrTS(Thr-51) (wild type), TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51), in which the value of kcat/KM for ATP in aminoacylation increases along the series. This is achieved by the value of KM decreasing significantly (2.5, 1.25, 0.29, and 0.019 mM, respectively) while there are smaller decreases in kcat (4.7, 4.0, 2.9, and 1.8 s-1, respectively). These variations cause each one of the enzymes to be more active than the others at particular concentrations of ATP. For example, at concentrations of ATP greater than 5.9 mM, TyrTS(Thr-51) is the most active, while TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51) are the most active at 5.9-2.2, 2.2-0.42, and less than 0.42 mM ATP, respectively. Interestingly, position 51 shows variation in tyrosyl-tRNA synthetases isolated from different organisms.     

Mutational switching of a yeast tRNA synthetase into a mammalian-like synthetase cytokine

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. A link was recently established between protein biosynthesis and cytokine signal transduction. Human tyrosyl-tRNA synthetase can be split into two fragments, each of which has a distinct cytokine function. This activity is specific to the human enzyme. It is absent in the enzymes from lower organisms such as bacteria and yeast. Here, yeast tyrosyl-tRNA synthetase (TyrRS), which lacks cytokine activity, was used as a model to explore how a human tyrosyl-tRNA synthetase during evolution acquired novel functions beyond aminoacylation. We found that a rationally designed mutant yeast TyrRS(ELR) gained cytokine function. The mutant yeast enzyme gained this function without sacrifice of aminoacylation activity. Therefore, relatively simple alteration of a basic structural motif imparts cytokine activity to a tRNA synthetase while preserving its canonical function. Further work established that mutational switching of a yeast protein to a mammalian-like cytokine was specific to this synthetase and not to just any yeast ortholog of a mammalian cytokine.     

Tyrosyl-tRNA synthetase from the bovine liver. Isolation and physico-chemical properties

Tyrosyl-tRNA synthetase of beef liver has been isolated and its properties have been studied. Tyrosyl-tRNA synthetase is a structural dimer of alpha 2 type. Mr of the enzyme subunit is about 59 kDa. Km values for substrates have been determined and compared with kinetic properties of tyrosyl-tRNA synthetases from different sources. The polymorphism of tyrosyl-tRNA synthetase was studied. The enzyme was separated into two different forms by chromatography on phosphocellulose P 11. P1-form is active only in the amino acid activation reaction. This form is not due to the phosphorylation of the enzyme. The low molecular weight form (38 kDa) was also isolated. This form appeared due to the limited endogenic proteolysis of the main form and retained full activity in the aminoacylation reaction. Tyrosyl-tRNA synthetase from beef liver has non-specific affinity to rRNA-sepharose.