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.

Effect of the overproduction of phenylalanyl- and threonyl-tRNA synthetases on tRNAPhe and tRNAThr concentrations in E. coli cells.

Transformation of an E. coli strain with a recombinant plasmid DNA (pB1) encoding the genes for phenylalanyl- and threonyl-tRNA synthetases causes overproduction of these enzymes by about 100- and 5-fold, respectively. A possible effect of the overproduction of the two aminoacyl-tRNA synthetases on intracellular cognate tRNA levels has been searched for by comparing tRNAThr and tRNAPhe aminoacylation capacities in the RNA extracts from strains carrying pB1 or pBR322 plasmid DNA. The answer is that the levels of these tRNAs are not changed by selective increase of the cognate synthetases.

Removal of the tightly bound zinc from Escherichia coli trypsin-modified methionyl-tRNA synthetase.

The study of the behaviour of Escherichia coli methionyl-tRNA synthetase with chelating agents has shown that only 1,10-phenanthroline has an inhibitory effect on the tRNAMet aminoacylation activity. Under identical buffer conditions the isotopic [32P]PPi-ATP exchange activity is insensitive. Dialysis of the enzyme against 1,10-phenanthroline causes a slow loss of zinc from the enzyme which is paralleled by an irreversible loss of both the aminoacylation and isotopic exchange activities. The loss of zinc becomes faster upon the addition of small amounts of guanidine hydrochloride to the dialysis buffer containing phenanthroline, presumably by partially unfolding the protein. Studies of the reversible denaturation of the enzyme by 5 M guanidine hydrochloride shows that the inclusion of EDTA produces an enzyme species that has lost both zinc and activity. The inactive apoenzyme prepared in guanidine and EDTA can regain activity by dilution in a zinc-containing buffer.

Proteolytic cleavage of methionyl transfer ribonucleic acid synthetase from Bacillus stearothermophilus: effects on activity and structure.

Methionyl-tRNA synthetase from Bacillus stearothermophilus, a dimer of molecular weight 2 X 85K, is converted by limited subtilisin digestion into a fully active monomeric fragment of molecular weight 64K. The reversible methionine activation reaction of these enzymes was followed through the variation of the intensity of their trypotophan fluorescence. Equilibrium and stopped-flow experiments show that the rate and mechanism for adenylate formation supported by the monomeric derivative are undistinguishable from those of each adenylating site of the native dimeric enzyme. In contrast, the rate of tRNA aminoacylation is improved upon limited proteolysis of the native enzyme. This behavior can be related to the anticooperativity of the binding of tRNA molecules to native dimeric enzyme. Accordingly, at 25 degrees C, the dimer might behave as a half-of-the-sites enzyme with only one active tRNA site at a time, compared to two after limited proteolysis with consequent irreversible disociation into two 64K fragments. Another modified form of the enzyme is obtained through limited tryptic digestion. This derivative is completely devoid of activity although its molecular weight under nondenaturating conditions remains undistinguishable from that of the 64K fragment generated by subtilisin. Denaturation reveals that this tryptic derivative is composed of two subfragments with molecular weights of 33K and 29K, respectively. The same fragments may also be directly obtained through limited tryptic digestion of the subtilsic fragment. Interestingly, although trypsin treatment has abolished the activity of the enzyme, fluorescence studies demonstrate that the ATP and methionine binding sites have remained intact. It is shown that the effect of the internal cut made by trypsin into the active 64K fragment has been to considerably depress the "coupling" between the methionine and nucleotide binding sites. Finally, the rate of inactivation of the enzyme by trypsin is observed to be substantially decreased by in situ synthetized methionyl adenylate but not by tRNA. These properties and others are discussed in relation to the problem of its significance of repeating sequences and structural "domains" within the class of aminoacyl-tRNA synthetases.

Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli.

Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II). It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site. The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.

Interaction of aspartate aminotransferase with mercurochrome. Relationship of an exposed thiol group of the enzyme to the active centre.

Mercurochrome strongly inhibits aspartate transaminase and 2,3-dicarboxyethylated aspartate transaminase. The native enzyme exhibits a biphasic time-course of inactivation by mercurochrome with second-order rate constants 1.62 x 10(4) M-1 - min-1 and 2.15 x 10(3) M-1 - min-1, whereas the modified enzyme is inactivated more slowly (second-order rate constant 6.1 x 10(2) M-1 - min-1) under the same conditions. The inhibitor inactivates native and modified enzyme in the absence as well as in the presence of substrates. Mercurochrome-transaminase interaction is accompanied by a red shift in the absorption maximum of the fluorochrome of about 10 nm. Difference spectra of the mercurochrome-enzyme system versus mercurochrome, compared with analogous spectra of mercurochrome-ethanol, revealed that the spectral shifts recorded during mercurochrome-transaminase interaction are similar to those that occur when mercurochrome is dissolved in non-polar solvents. Studies of mercurochrome complexes with native or modified transaminase, isolated by chromatography on Sephadex G-25, revealed that native transaminase is able to conjugate with four mercurochrome molecules per molecule, but the modified enzyme is able to conjugate with only two mercurochrome molecules per molecule.