Analysis of protein sequences and protein complexes by matrix-assisted laser desorption/ionization mass spectrometry.

In the context of proteome analysis, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) can fulfil the two tasks of primary structure verification and protein identification. As an illustration of the first of these tasks, the sequence of Eschericha coli isoleucyl-tRNA synthetase, a protein with 15 reported sequence conflicts, has been established by means of MALDI mass mapping. The identification of mitochondrial proteins participating in a yeast supramolecular complex exhibiting NADH dehydrogenase activity highlights the performances of MALDI-MS for the second task. The spectral suppression phenomenon occurring for complex peptide mixtures analysed by MALDI is discussed, as well as the role of post-source decay analysis for confident protein identification.

Sites of phosphorylation by protein kinase A in CDC25Mm/GRF1, a guanine nucleotide exchange factor for Ras.

Activation of the neuronal Ras GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 is known to be associated with phosphorylation of serine/threonine. To increase our knowledge of the mechanism involved, we have analyzed the ability of several serine/threonine kinases to phosphorylate CDC25Mm in vivo and in vitro. We could demonstrate the involvement of cAMP-dependent protein kinase (PKA) in the phosphorylation of CDC25Mm in fibroblasts overexpressing this RasGEF as well as in mouse brain synaptosomal membranes. In vitro, PKA was found to phosphorylate multiple sites on purified CDC25Mm, in contrast to protein kinase C, calmodulin kinase II, and casein kinase II, which were virtually inactive. Eight phosphorylated serines and one threonine were identified by mass spectrometry and Edman degradation. Most of them were clustered around the Ras exchanger motif/PEST motifs situated in the C-terminal moiety (residues 631-978) preceding the catalytic domain. Ser745 and Ser822 were the most heavily phosphorylated residues and the only ones coinciding with PKA consensus sequences. Substitutions S745D and S822D showed that the latter mutation strongly inhibited the exchange activity of CDC25Mm on Ha-Ras. The multiple PKA-dependent phosphorylation sites on CDC25Mm suggest a complex regulatory picture of this RasGEF. The results are discussed in the light of structural and/or functional similarities with other members of this RasGEF family.

Valyl-tRNA synthetase from Escherichia coli MALDI-MS identification of the binding sites for L-valine or for noncognate amino acids upon qualitative comparative labeling with reactive amino-acid analogs.

Bromomethyl ketone derivatives of L-valine (VBMK), L-isoleucine (IBMK), L-norleucine (NleBMK) and L-phenylalanine (FBMK) were synthesized. These reagents were used for qualitative comparative labeling of Escherichia coli valyl-tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in order to identify the binding sites for L-valine or noncognate amino acids. Labeling of E. coli ValRS with the substrate analog valyl-bromomethyl ketone (VBMK) resulted in a complete loss of valine-dependent isotopic [32P]PPi-ATP exchange activity. L-Valine protected the enzyme against inactivation. Noncognate amino acids analogs isoleucyl-, norleucyl- and phenylalanyl-bromomethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the activity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix-assisted laser desorption-ionization mass spectrometry designated cysteines 424 and 829 as the target residues of the substrate analog VBMK on E. coli ValRS, whereas, altogether, IBMK, NleBMK and FBMK labeled His266, Cys275, His282, His433 and Cys829, of which Cys275, His282 and His433 were labeled in common by all three noncognate amino-acid-derived bromomethyl ketones. With the exception of Cys829, which was most likely unspecifically labeled, the amino-acid residues labeled by the reagents derived from noncognate amino acids were distributed between two fragments 259-291 and 419-434 in the primary structure of E. coli ValRS. In fragment 419-434, Cys424 was specifically labeled by the substrate analog VBMK, while His433 was labeled in common by all the used bromomethyl ketone derivatives of noncognate amino acids, suggesting that the synthetic site where aminoacyl adenylate formation takes place on E. coli ValRS is built up of two subsites. One subsite containing Cys424 might represent the catalytic locus of the active center where specific L-valine activation takes place. The second subsite containing His433 might represent the binding site for noncognate amino acids. The fact that Cys275 and His282, fragment 259-291, were labeled by IBMK, NleBMK and FBMK, but not by the substrate analog VBMK, suggests that these residues might be located at or near the editing site of E. coli ValRS. Comparison of fragment 259-291 with all the available ValRS amino-acid sequences revealed that His282 is strictly conserved, with the exception of its replacement by a glycine in a subgroup corresponding to the archaebacteria. Because a nucleophile is needed in the editing site to achieve hydrolysis of an undesired product at the level of the carbonyl group thereof, it is proposed that the conserved His282 of E. coli ValRS is involved in editing.

Enzyme-induced covalent modification of methionyl-tRNA synthetase from Bacillus stearothermophilus by methionyl-adenylate: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.

Methionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was shown to undergo covalent methionylation by a donor methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself. Covalent reaction of methionyl-adenylate with the synthetase or other proteins proceeds through the formation of an isopeptide bond between the carboxylate of the amino acid and the epsilon-NH2 group of lysyl residues. The stoichiometries of labeling, as followed by TCA precipitation, were 2.2 +/- 0.1 and 4.3 +/- 0.1 mol of [14C]Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B. stearo methionyl-tRNA synthetase, respectively. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-261, -295, -301 and -528 (or -534) of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. By analogy with the 3D structure of the monomeric M547 species of E. coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be located in the catalytic crevice of the thermostable enzyme where methionine activation and transfer take place. It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl residues.

Transfer RNA-pseudouridine synthetase Pus1 of Saccharomyces cerevisiae contains one atom of zinc essential for its native conformation and tRNA recognition.

RNA:pseudouridine synthetase (Pus1) from Saccharomyces cerevisiae is a multisite specific enzyme that catalyzes the formation of pseudouridine at positions 34 and 36 of intron-containing precursor tRNAIle and at positions 27 and/or 28 of several yeast tRNAs. In this paper we demonstrate that the purified recombinant Pus1, expressed in Escherichia coli, contains one atom of zinc per 63-kDa monomer, as determined by atomic absorption spectroscopy. This zinc ion could not be removed by treatment with EDTA or urea. However, a zinc-depleted enzyme was obtained after prolonged dialysis against the specific chelating agent 1,10-phenanthroline. Removal of the zinc ion resulted in inactivation of the enzyme with concomitant loss of its ability to bind tRNA. Dialysis of the zinc-depleted inactive enzyme against buffer containing zinc ions led to recovery of up to 25% of bound zinc in parallel with 25% of its initial activity. Removal of the tightly bound zinc atom resulted in a conformational change of the protein, as determined by analytical ultracentrifugation, with minor changes in the internal structure of the protein, as evidenced by circular dichroism and infrared and fluorescence spectroscopy. Our results are consistent with a structural role for the zinc in the tRNA-pseudouridine synthetase Pus1; zinc ion could maintain the association between domains structurally organized around the coordinated metal ion. Zinc chelation was never demonstrated for any of the tRNA-pseudouridine synthetases characterized so far.

Covalent methionylation of Escherichia coli methionyl-tRNA synthethase: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.

Methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by methionyl-tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the epsilon-NH2 group of lysyl residues. It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl-adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Following the incorporation of 7.0 +/- 0.5 mol of methionine per mol of a monomeric truncated methionyl-tRNA synthetase species, the enzymic activities of [32P]PPi-ATP isotopic exchange and tRNA(Met) aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNA(Met) protected the enzyme against inactivation and methionine incorporation. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-114, -132, -142 (or -147), -270, -282, -335, -362, -402, -439, -465, and -547 of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. These lysyl residues are distributed at the surface of the enzyme between three regions [114-150], [270-362], and [402-465], all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA.

Affinity labeling of Escherichia coli histidyl-tRNA synthetase with reactive ATP analogues. Identification of labeled amino acid residues by matrix assisted laser desorption-ionization mass spectrometry.

Recent affinity labeling studies have revealed that dimeric histidyl-tRNA synthetase from Escherichia coli displayed half-of-the-sites reactivity toward labeling with pyridoxal 5'-phosphate [Kalogerakos, T., Hountondji, C., Berne, P. F., Dutka, S. & Blanquet, S. (1994) Biochimie (Paris) 76, 33-44]. In the present report, affinity labeling studies were conducted by using other ATP analogues such as pyridoxal 5'-diphospho-5'-adenosine (pyridoxal-ppAdo), pyridoxal 5'-triphospho-5'-adenosine (pyridoxal-pppAdo), pyridoxal 5'-diphosphate (pyridoxal-P2) and 5'-p-fluorosulfonylbenzoyladenosine (FSO2BzAdo). The histidine-dependent isotopic [32P]PP/ATP exchange activity of His-tRNA synthetase was rapidly and completely lost upon incubation with either pyridoxal-ppAdo, pyridoxal-pppAdo or pyridoxal-P2, followed by reduction with sodium borohydride. Complete inactivation of His-tRNA synthetase corresponded to the incorporation of 2.8 mol of either pyridoxal-ppAdo or pyridoxal-P2/mol dimeric synthetase. Incubation of His-tRNA synthetase with FSO2BzAdo also resulted in a complete inactivation of the synthetase. However, contrasting with the pyridoxal derivatives, the plot of the residual enzymatic activity against the amount of covalently bound FSO2BzAdo appeared biphasic. In the early stages of inactivation, the relationship between the amount of residual activity and FSO2BzAdo incorporation was linear and extrapolated to a stoichiometry of 1.1 mol reagent/mol His-tRNA synthetase, suggesting that the labeling of one subunit was sufficient to inactivate one dimeric His-tRNA synthetase molecule. At longer incubation periods, additional reagent incorporation occurred and culminated at 2.5 mol label/mol His-tRNA synthetase. Excess of MgATP protected the enzyme against inactivation by either studied reagent. The labeled amino acid residues were identified by matrix-assisted-laser-desorption-ionization mass spectrometry, by measuring the peptide mass increase caused by the reagents. An identical set of four lysyl residues (Lys2, Lys118, Lys369 and Lys370 of His-tRNA synthetase) was found attached to pyridoxal-ppAdo or pyridoxal-P2. In addition, pyridoxal-ppAdo labeled the alpha-amino group of the N-terminal alanine. In a His-tRNA synthetase sample having incorporated 2.5 mol FSO2BzAdo/mol), the labeled amino acid residues were Lys118, Lys196, Tyr262 (or Tyr263), Lys369 and Lys377. Whatever the used reagent, Lys118 appeared to be the predominantly labeled residue, Lys118 belongs to fragment 112-124 (RHERPQK-GRYRQF) corresponding to motif 2 of class 2 aminoacyl-tRNA synthetases. The other modified lysyl residues (lysines 369, 370 and 377) are close to the catalytic motif 3, in the C-terminal region of the synthetase. Tyr262 and Tyr263 belong to a fragment 256-263 (LVRGLDYY) highly conserved among all known His-tRNA synthetase primary structures. Examination of the recently solved structure of crystalline E. coli His-tRNA synthetase [Amez, J. G., Harris, D. C., Mitschler, A., Rees, B., Francklyn, C. S. & Moras, D. (1995) EMBO J. 14, 4143-4155] shows that, with the exception of lysines 369, 370 and 377, the location of which may account for peculiar accessibility and reactivity, all the amino acid residues identified in this study map near the enzyme nucleotide-binding site, at the N-terminal catalytic domain of the synthetase.

Affinity labeling of the two species of Escherichia coli lysyl-tRNA synthetase with adenosine di- and triphosphopyridoxals.

Lysyl-tRNA synthetase (LysRS), a representative of the class 2 aminoacyl-tRNA synthetases, occurs as two species in Escherichia coli: LysRSs and LysRSu. To identify the ATP-binding site in this enzyme, we have applied affinity labeling with reactive adenine nucleotide analogs. Incubation of either enzyme species with adenosine di- or triphosphopyridoxal, followed by borohydride reduction, resulted in a time-dependent incorporation of the reagent, accompanied with the loss of both tRNA(Lys) aminoacylation, and lysine-dependent isotopic ATP-PPi exchange activities. LysRSu appeared less sensitive to adenosine triphosphopyridoxal than LysRSs. Complete inactivation with either reagent corresponded to the incorporation of about 2 mol of reagent per mol of dimeric enzyme. MgATP and ATP protected both enzyme species against the inactivation, suggesting that the modification occurs at the ATP-binding site. Sequence analysis of the labeled peptide isolated from the inactivated LysRSs and LysRSu revealed that bulk of the label was distributed among six lysyl residues at positions 25, 82, 114, 156, 364, and 505, with preference for Lys-114 and Lys-156. In LysRSs, Lys-132 and Lys-185 were also modified by both reagents, although these residues are not conserved in LysRSu. It is concluded that the folding of the LysRSs and LysRSu polypeptides and the relative locations of the identified lysyl residues with respect to the binding site for the two labels are very similar.

Affinity labeling of Escherichia coli lysyl-tRNA synthetase with pyridoxal mono- and diphosphate.

Pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphosphate (PLDP) were used to identify lysyl residues at the phosphate-binding locus in the lysS-encoded and the lysU-encoded lysyl-tRNA synthetases (LysRSs and LysRSu, respectively) from Escherichia coli. Incubation of LysRSs with either reagent, followed by borohydride reduction, resulted in a time-dependent covalent incorporation of the reagent, accompanied with the loss of both tRNA(Lys) aminoacylation and lysine-dependent ATP-PPi exchange activities. By contrast, LysRSu activity was insensitive to prolonged incubation with either reagent, possibly reflecting a difference at the phosphate-binding locus in the two enzyme species. MgATP protected LysRSs against inactivation by PLP or PLDP. Complete inactivation of LysRSs corresponded to the incorporation of 2.6 +/- 0.1 mol of PLP or PLDP per mol of dimeric enzyme. Either reagent was found to label the same set of eight lysyl residues (Lys-25, Lys-82, Lys-114, Lys-132, Lys-156, Lys-185, Lys-364, and Lys-505) as adenosine di- or triphosphopyridoxal (see the preceding paper in this issue). These lysyl residues might represent the subsite for the phosphate moiety of ATP in LysRSs. None of the identified lysyl residues is located within the three sequence motifs considered as characteristic of the class 2 aminoacyl-tRNA synthetases. The present results are discussed on the basis of the crystalline structure of the closely related aspartyl-tRNA synthetase from Saccharomyces cerevisiae.

Modification of aminoacyl-tRNA synthetases with pyridoxal-5'-phosphate. Identification of the labeled amino acid residues.

The isotopic [32P]PPi-ATP exchange activity of isoleucyl-, valyl-, histidyl-, tyrosyl- and methionyl-tRNA synthetases from Escherichia coli are lost upon incubation in the presence of pyridoxal-5'-phosphate (PLP). When the residual activity of either isoleucyl-, valyl- or methionyl-tRNA synthetase (monomeric truncated form) was plotted as a function of the number of PLP molecules incorporated per enzyme molecule, the plots obtained appeared biphasic. Below 50% inactivation of these enzymes, PLP incorporation varied linearly with the isotopic exchange measurements, and extrapolation of the first half of the plot indicated a stoichiometry of 1.10 +/- 0.05 mol of PLP incorporated per mol of 100% inactivated synthetase. Beyond 50% inactivation, the graph deviated from its initial slope, and up to 4-5 mol of PLP were incorporated per mol of synthetase at the highest used PLP concentrations. In the cases of homodimeric histidyl- and tyrosyl-tRNA synthetases, extrapolation of the graph at 100% inactivation indicated 2.8 +/- 0.1 and 2.4 +/- 0.1 mol of PLP incorporated per mol of enzyme, respectively. PLP-labeled peptides were obtained through trypsin digestion and RPLC purification, prior to Edman degradation analysis. PLP-labeled residues were identified as lysines 132, 332, 335 and 402 of monomeric methionyl-tRNA synthetase, lysines 332, 335, 402, 465, 596 and 640 of native dimeric methionyl-tRNA synthetase, lysines 22, 117, 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557, 559, 593 and 909 of valyl-tRNA synthetase, lysines 2, 118, 369 and 370 of histidyl-tRNA synthetase, and lysine 237 of tyrosyl-tRNA synthetase. In addition, the amino terminal residue of the polypeptide chain(s) of either isoleucyl-, valyl-, histidyl- or methionyl-tRNA synthetases was found labeled. Among these residues, lysines 332, 335 and 402 of monomeric methionyl-tRNA synthetase as well as lysines 332, 335, 402 and 596 of dimeric methionyl-tRNA synthetase, lysines 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557 and 559 of valyl-tRNA synthetase, lysines 2, 369 and 370 of histidyl-tRNA synthetase, and lysine 237 of tyrosyl-tRNA synthetase were labeled in the presence of PLP concentrations smaller than or equal to 1 mM, and are shown to be critical for the activity of the enzymes. It is concluded that these residues participate to the binding sites of the phosphates of ATP on the studied synthetases.

Interaction of the tRNA(Phe) acceptor end with the synthetase involves a sequence common to yeast and Escherichia coli phenylalanyl-tRNA synthetases.

Modified lysines resulting from the cross-linking of the 3' end of tRNA(Phe) to yeast phenylalanyl-tRNA synthetase (an enzyme with an alpha 2 beta 2 structure) have been characterized by sequencing the labeled chymotryptic peptides that were isolated by means of gel filtration and reversed-phase chromatography. The analysis showed that Lys131 and Lys436 in the alpha subunit are the target sites of periodate-oxidized tRNA(Phe). Mutant protein with a Lys----Asn substitution established that each lysine contributes to the binding of the tRNA but is not essential for catalysis. The major labeled lysine (K131) belongs to the sequence IALQDKL (residues 126-132), which shares three identities with the peptide sequence ADKL found around the tRNAox-labeled Lys61 in the large subunit of Escherichia coli phenylalanyl-tRNA synthetase [Hountondji, C., Schmitter, J. M., Beauvallet, C., & Blanquet, S. (1987) Biochemistry 26, 5433-5439].

Affinity labeling of aminoacyl-tRNA synthetases with adenosine triphosphopyridoxal: probing the Lys-Met-Ser-Lys-Ser signature sequence as the ATP-binding site in Escherichia coli methionyl-and valyl-tRNA synthetases.

Pyridoxal 5'-triphospho-5'-adenosine (AP3-PL), the affinity labeling reagent specific for lysine residues in the nucleotide-binding site of several enzymes [Tagaya, M., & Fukui, T. (1986) Biochemistry 25, 2958-2964; Yagami, T., Tagaya, M., & Fukui, T. (1988) FEBS Lett. 229, 261-264], was used to identify the ATP-binding site of Escherichia coli methionyl-tRNA synthetase (MetRS). Incubation of this enzyme with AP3-PL followed by reduction with sodium borohydride resulted in a rapid inactivation of both the tRNA(Met) aminoacylation and the methionine-dependent ATP-PPi exchange activities. Complete inactivation corresponded to the incorporation of 0.98 mol of AP3-PL/mol of monomeric trypsin-modified MetRS. ATP or MgATP protected the enzyme from inactivation. The labeling with AP3-PL was also applied to E. coli valyl-tRNA synthetase (ValRS). Both the tRNA(Val) aminoacylation and the valine-dependent ATP-PPi exchange activities were abolished by the incorporation of 0.91 mol of AP3-PL/mol of monomeric ValRS. AP3-PL was found attached to lysine residues 335, 402, and 528 in the primary structure of MetRS. In the case of ValRS, the AP3-PL-labeled residues corresponded to lysines 557, 593, and 909. We therefore conclude that these lysines of MetRS and ValRS are directed toward the ATP-binding site of these synthetases, more specifically at or close to the subsite for the gamma-phosphate of ATP. AP3-PL-labeled Lys-335 of MetRS and Lys-557 of ValRS belong to the consensus tRNA CCA-binding Lys-Met-Ser-Lys-Ser sequence [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of the active site of Escherichia coli methionyl-tRNA synthetase: identification of amino acid residues labeled by periodate-oxidized tRNA(fMet) molecules having modified lengths at the 3'-acceptor end.

Initiator tRNA molecules modified at the 3'-end and lacking either the A76 (tRNA-C75), the C75-A76 (tRNA-C74), the C74-C75-A76 (tRNA-A73), or the A73-C74-C75-A76 (tRNA-A72) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTST), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C75ox, tRNA-C74ox, tRNA-A73ox, and tRNA-A72ox) abolished both the isotopic [32P]PPi-ATP exchange and the tRNA aminoacylation activities of the enzyme. In the presence of limiting concentrations of the various tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase. Specificity of the labeling was further established by demonstrating that tRNA-C75ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C75. The peptides of MTST labeled with either tRNA-C75ox or tRNA-C74ox were identified. The chymotryptic digestion of the covalent MTST.[14C]tRNA-C75ox complex yielded four peptides (A-D). In the case of tRNA-C74ox, only two of the above peptides (C and D) were identified. Peptides A, B, C, and D corresponded to fragments Ser334-Phe340, Lys61-Leu65, Val141-Tyr165, and Glu433-Phe437, respectively, in the MTST primary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Methionyl-tRNA synthetase from E. coli--a review.

Methionyl-tRNA synthetase (MetRS) from E coli is a dimer composed of 2 identical subunits of Mr 76 kDa. A fully active monomeric fragment (64 kDa) could be obtained by mild proteolysis of the native dimer. Earlier studies reviewed in Blanquet et al (1979) have compared the catalytic mechanisms of native and trypsin-modified MetRS. Moreover, the truncated form of the enzyme was crystallized and its 3-D structure solved at low resolution. In the last few years, the availability of the corresponding metG gene has facilitated the development of studies using affinity labelling and site-directed mutagenesis techniques. In parallel, the 3-D structure has been solved at a resolution of 2.5 A. These convergent approaches have allowed significant progress in the understanding of the structure-function relationships of this enzyme, and, in particular, of the rules governing the recognition of tRNA.

Analytical strategy for determination of active site sequences in aminoacyl-tRNA synthetases.

Affinity labelling with radioactive, periodate-oxidized tRNA has been used to investigate the structures of tRNA-binding sites in Escherichia coli aminoacyl-tRNA synthetases. Labelled peptides were isolated by means of a combination of techniques involving chymotryptic digestion of the enzyme, gel filtration, ribonuclease digestion of tRNA, chromatography on a TSK 2000 column and reversed-phase chromatography. An isocratic phenylthiohydantoin identification system has been interfaced to a sequencer, allowing the characterization of modified lysine residues by means of both chromatographic retention and liquid scintillation counting.

Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe.

Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[14C]tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe). The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the alpha subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].

Sequence similarities among the family of aminoacyl-tRNA synthetases.

Recent affinity labeling studies have led to the identification of lysine residues at the CCA binding site of tRNA in Escherichia coli methionyl- and tyrosyl-tRNA synthetases. The comparison of the labeled peptides to the known primary structures of the aminoacyl-tRNA synthetases reveals new sequence similarities among this family of enzymes. These similarities include a 'constant' lysine residue whose functional significance is discussed. Moreover, a systematic computer analysis was conducted to search for similarities between the aminoacyl-tRNA synthetases taken as pairs.

Escherichia coli tyrosyl- and methionyl-tRNA synthetases display sequence similarity at the binding site for the 3'-end of tRNA.

Covalent modification of Escherichia coli tyrosyl-tRNA synthetase (TyrRS) by the 2',3'-dialdehyde derivative of tRNATyr (tRNAox) resulted in a time-dependent inactivation of both ATP-PPi exchange and tRNA aminoacylation activities of the enzyme. In parallel with the inactivation, covalent incorporation of approximately 1 mol of [14C]tRNATyrox/mol of the dimeric synthetase occurred. Intact tRNATyr protected the enzyme against inactivation by the tRNA dialdehyde. Treatment of the TyrRS-[14C]tRNATyr covalent complex with alpha-chymotrypsin produced two labeled peptides (A and B) that were isolated and identified by sequence analysis. Peptides A and B are adjacent and together span residues 227-244 in the primary structure of the enzyme. The three lysine residues in this sequence (lysines-229, -234, and -237) are labeled in a mutually exclusive fashion, with lysine-234 being the most reactive. By analogy with the known three-dimensional structure of the homologous tyrosyl-tRNA synthetase from Bacillus stearothermophilus, these lysines should be part of the C-terminal domain which is presumed to bind the cognate tRNA. Interestingly, the labeled TyrRS structure showed significant similarities to the structure around the lysine residue of E. coli methionyl-tRNA synthetase which is the most reactive toward tRNAMetf(ox) (lysine-335) [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180].

Methionyl-tRNA synthetase from Escherichia coli: primary structure at the binding site for the 3'-end of tRNAfMet.

It was previously shown that when the tryptic fragment of methionyl-tRNA synthetase from Escherichia coli is incubated with periodate-treated initiator tRNA, it is inactivated due to the formation of a covalent 1:1 complex that could be stabilized by reduction with cyanoborohydride [Hountondji, C., Fayat, G., & Blanquet, S. (1979) Eur. J. Biochem. 102, 247-250]. In this work, the residues labeled in the trypsin-modified enzyme have been identified. After chymotryptic digestion of the protein-tRNA complex, two major labeled peptides (A and B) and a minor one (C) were isolated and identified by sequencing. The radioactivity associated with peptides A-C represented 65-75, 20-25, and 2-4%, respectively, of the radioactivity eluted from the peptide maps. Peptides A and B encompassed lysines-335 and -61, respectively. Both these lysines were fully labeled. Peptide C encompassed lysines-142, -147, and -149, each of which was incompletely labeled. The significance of these results is discussed in light of the known crystallographic structure of the enzyme.