The Mechanism of Action of Methionyl‐tRNA Synthetase from Escherichia coli

The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethy1eneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses of adenosine(5')tetraphospho(5')adenosine (Ap,A) and adenosine(5')triphospho(5')adenosine (Ap,A), also catalyzes the formation of ADP from inorganic phosphate and the enzyme-bound glycyl adenylate. Accordingly it was shown that E. coli glycyl-tRNA synthetase, in the presence of inorganic phosphate, glycine, and Mg2 and Ap,A. It was furthermore demonstrated that the only pathway by which a synthetase-catalyzed degradation of Ap,A can occur is through the reaction between inorganic phosphate and the enzyme-bound glycyl adenylate, synthesized from Ap,A. Likewise a 20-fold increase of the phosphorolytic activity of the investigated synthetase was observed when Mg2+ was replaced by Mn2+.Besides establishing the phosphorolytic activity of the applied enzyme, the study also showed that the preparation catalyzes a glycine-independent transfer of the y-phosphate group from ATP to nucleoside 5'-diphosphates.The importance of the observed reaction between inorganic phosphate and enzymebound aminoacyl adenylate in relation to the remaining catalytic activities of aminoacyl-tRNA synthetases is discussed, as well as the biological significance of the reaction.

Section: Discussionsupporting

confidence: 89%

Phosphorolytic activity of Escherichia coli glycyl‐tRNA synthetase towards its cognate aminoacyl adenylate detected by ³¹P‐NMR spectroscopy and thin‐layer chromatography

Led

Switon

Jensen

1983

“…It has been shown that this enzyme is functionally a dimer having two binding sites for methionine and methioninyl adenylate [55] and two for tRNAMet [56]. However, equilibrium dialysis experiments show that there are four distinct binding sites for ATP, only two of which are catalytic sites involved in methionyl adenylate formation (Fayat, G., Waller, J. P., unpublished results); it is these two sites which have been directly examined in the present study.…”

Section: Modifications To the Ribosementioning

confidence: 74%

“…However, the cishydroxyl arrangement is not essential, for the ribose can be replaced by arabinose (compound36), containing trans-hydroxyls, with only a seven-fold increase in inhibition constant. It is not surprising to observe that L-adenosine [56], the enantiomer of the naturally occurring adenosine, is not an inhibitor. This compound contains all the groups necessary for recognition, but the considerable change in their spatial distribution places them in positions not available for efficient binding to the enzyme.…”

Section: Modifications To the Ribosementioning

confidence: 99%

The Effect of Adenosine Analogues on the ATP‐Pyrophosphate Exchange Reaction Catalysed by Methionyl‐tRNA Synthetase

Lawrence

Shire

Waller

1974

Self Cite

Following the finding that the ATP-PPi exchange reaction and the aminoacylation of tRNA catalysed by methionyl-tRNA synthetase from Escherichia coli are inhibited by adenosine, numerous analogues of adenosine have been examined to determine the nature of the specificity. The main features observed are as follows.1. Modifications to positions 1, 2 and 6 of the purine ring of adenosine largely eliminate the inhibition of the ATP-pyrophosphate exchange reaction.2. The replacement of the nitrogen a t position 7 with a carbon has virtually no effect. 3. Substitutions a t positions 8 give analogues exhibiting a wide range of inhibition constants; 8-aminoadenosine is a highly effective inhibitor, with a Ki 300 times lower than that of adenosine.4. The sugar part of the nucleoside is essential for binding, but can be considerably modified without too great a detrimental effect, particularly in the 5'-position.5. For the nucleosides which have a low inhibition constant the presence of a triphosphate group lowers the affinity, but for the analogues exhibiting a high inhibition constant a triphosphate group has no effect.The amino-acid sequences a t the active sites of the aminoacyl-tRNA synthetases remain to be determined as also do the functional groups which account for their catalytic activity. I n the absence of such information much can be learned from studies of substrate interactions. In addition, these studies could lead to the design of prospective affinity labels, which would be invaluable tools for both sequence analysis and for the elucidation of the mechanisms, of action of these important enzymes. It is clear from the few reports available that, apart from the fact that all the activating enzymes utilise ATP as a substrate, their active sites differ considerably in their ability to recognize modified substrates and inhibitors.During our study of the methionyl-tRNA synthetase vie have found that adenosine is a competitive inhibitor with respect to ATP of the ATP-PPi exchange reaction catalyzed by this enzyme [l].The nucleoside also inhibits the pyrophosphate exchangc reaction catalyzed by other activating enzymes [l] and adenosine and analogues of adenosine have been used to investigate the ATP binding site of phenylalanyl-tRNA and tyrosyl-tRNA synthetases [2-41. This paper reports the effect of many structural analogues of adenosine on the ability of Enzyme. AIethionyl-tRNA synthetase or L-methionine : tRNA ligase (AMP) (EC 6.1.1.10).purified methionyl-tRNA synthetase to catalyse the ATP-PPi exchange reaction. MATERIALS AND METHODS2-Trifluoromethyladenosine, 2-methylthioadenosine and 2-methoxyadenosine were kindly donated by Dr H. Maguire (University of Sydney). 6-Hydroxyethyladenosine was obtained after phosphorylase digestion of the 6-hydroxyethyladenosine-5'-diphosphate provided by Dr A. Michelson (Paris). 8-Bromoadenosine was a gift from Dr J. Catlin (Saclay).5'-0-Carboxymethyladenosine and 5'-O-hydroxyethyladenosine were donated by Dr S. David (Orsay).5'-S-Adenosyl 3-thiopropionic acid, 5'-S-isobutylade...

“…The HDO signal in the 'H-NMR spectrum was thus kept at a minimum. Concentrations of the enzymes and the nucleotides were measured spectrophotometrically using the data: A2mggi;'' = 1.39 cm-' (methionyl tRNA synthetase; molecular mass 152 kDa [30], 1.72 cm-' (truncated methionyl tRNA synthetase, molecular mass 64 kDa) and 0.54 cm-' (pyruvate kinase; molecular mass 238 kDa) [31], and AyF9 = 15.4 cm-for ATP. Pyruvate kinase is a tetramer with one catalytic MgATP binding site/subunit.…”

Section: Nmr Samplesmentioning

confidence: 99%

Conformation of MgATP bound to nucleotidyl and phosphoryl transfer enzymes ¹H‐transferred NOE measurements on complexes of methionyl tRNA synthetase and pyruvate kinase

Landy

Ray

Plateau

et al. 1992

The conformations of MgATP bound to a nucleotidyl transfer enzyme, methionyl tRNA synthetase and a phosphoryl transfer enzyme, pyruvate kinase, were studied by transferred NOE (TRNOE) measurements in 'H NMR. The experiments were performed on D 2 0 solutions at 276 MHz and 300 MHz, and 10°C in the presence of approximately a tenfold excess of substrate over the enzyme (sites). Selective inversion of chosen resonances was accomplished with an appropriately tailored DANTE sequence consisting of 100 phase-alternating hard 1.8 O pulses. NOE measurements were made in terms of difference spectra (with and without inversion) at 6-8 delay times ranging from 10 -500 ms following the DANTE sequence. A full complement of ten NOE build-up curves obtained for each enzyme complex was analyzed by using the complete relaxation-matrix method (which includes all the non-exchangeable protons in MgATP) suitably modified to include exchange between bound and free substrate. Molecular mechanics computations were used to examine the energetic implications of the NOE-determined structure. The final structures obtained for MgATP bound to the two enzymes were very similar to each other, with a 3'-endo sugar pucker and an anti conformation with a glycosidic torsional angle (0; -C\ -N9 -C,) of 39" & 4". Both enzymes contain multiple binding sites for MgATP and hence the structure obtained in each case represents an average due to chemical exchange. However, TRNOE experiments performed on a tryptic fragment of methionyl tRNA synthetase which has a single MgATP binding site, show that the same structure fits these measurements as well. This evidence, coupled with the striking similarity of the structures deduced, for the two enzyme complexes, and the reciprocal sixth-power dependence of NOE on interproton distance, strongly suggests that the conformations at the individual binding sites of both the enzymes are virtually identical. This conclusion is in contrast with multiple conformations of MgATP bound to pyruvate kinase, proposed by Rosevear, P. R., Fox, T. L. & Mildvan, A. S . (1987) Biochemistry 26, 3487 -3493 Enzymes for which ATP is a substrate occur in a variety of biochemical pathways. These enzymes fall into three categories on the basis of the point of cleavage in the phosphate chain of ATP in the reaction: phosphoryl transfer, nucleotidyl transfer and pyrophosphoryl transfer. All three categories of enzymes require a divalent cation, Mg(II), as an obligatory component in the reaction. In the general area of inquiring into the molecular basis of enzyme catalysis by characterizing structures of enzyme -substrate complexes, the .4TP-utilizing enzymes offer the scope of comparing such structures for the three categories of enzymes that use the same substrate but catalyze cleavage at different places in the molecule. The question as to which structural features differentiate the enzyme -nucleotide complexes of phosphoryl transfer enzymes from nucleotidyl transfer enzymes, in Correspondence to B.