Aminoacyl Adenylate Research Articles

Gramicidin S synthetase. Temperature dependence and thermodynamic parameters of substrate amino acid activation reactions.

In the biosynthesis of the cyclic decapeptide antibiotic gramicidin S, the constituent amino acids are activated by a two-step mechanism involving aminoacyl adenylate and thio ester formation which are both reversible processes. The dissociation constants (KD) for the gramicidin S synthetase-substrate amino acid-thio ester complexes are 100-1000-fold lower compared to the KM data of the preceding aminoacyl adenylate reactions. The affinity for these substrates is appreciably higher at the thio template sites than at the aminoacyl adenylate reaction centers. Therefore, the activation equilibria are quantitatively shifted toward thio ester formation. A set of thermodynamic parameters for the activation processes was determined from the temperature dependence of the KM and KD data. Reaction enthalpies were obtained from a van't Hoff analysis of these constants. delta G degree for the substrate activation reactions of the heavy enzyme of gramicidin S synthetase (GS 2) is predominantly controlled by entropy contributions. In contrast, the overall activation and concomitant racemization of phenylalanine by phenylalanine racemase (GS 1) are exothermic processes which are distinguished by a small negative reaction entropy.

A peculiar property of aspartyl-tRNA synthetase from bakers' yeast: chemical modification of the protein by the enzymically synthesized aminoacyl adenylate

A peculiar property of aspartyl-tRNA synthetase from bakers' yeast: chemical modification of the protein by the enzymically synthesized aminoacyl adenylate

Reactive sulfhydryl groups involved in the aminoacyl adenylate activation reactions of the gramicidin S synthetase 2.

Six accessible sulfhydryl groups of the light component of gramicidin S synthetase (GS 1) were titrated with N-ethylmaleimide (Ma1NEt) as well as 3,3'-dithiobis(6-nitrobenzoic acid) (Nbs2). Twenty-four thiols were detected in the heavy enzyme (GS 2) using Ma1NEt as the modifier. Substrate amino acid-induced protection of GS 2 against deactivation by Ma1NEt indicates that in addition to the specific thiols at the thiotemplates of gramicidin S synthetase reactive SH groups are also involved in the primary aminoacyl adenylate activation reactions. Obviously 2 sulfhydryl groups are essential for the adenylation of each L-Pro, L-Val and L-Leu, while 3 thiols were detected for the ornithine activation. Agents like Ma1NEt or Nbs2 inhibit the thiolation of the substrate amino acids of gramicidin S synthetase and gramicidin S formation more severely than the aminoacyl adenylate activation reactions which are affected at 10-100-fold higher inhibitor concentrations. These processes can be studied separately if the thioester formation sites are inhibited at low concentrations of sulfhydryl inhibitors.

Fast kinetic study of yeast phenylalanyl-tRNA synthetase: role of tRNAPhe in the discrimination between tyrosine and phenylalanine.

An extensive study of the discrimination between phenylalanine and tyrosine by yeast phenylalanyl-tRNA synthetase was carried out in the presence of native tRNAPhe. Our results clearly show that, besides the previously reported dissociation of tyrosyl adenylate from the enzyme template [Lin, S. X., Baltzinger, M., & Remy, P. (1983) Biochemistry 22, 681-689], two more correction processes are involved in the rejection of tyrosine in the presence of tRNAPhe. A minor part of the misactivated tyrosine is indeed transferred to tRNAPhe, but the resulting misaminoacylated tRNA is very rapidly hydrolyzed (kh approximately equal to 60 s-1), as it has already been shown for other systems. However, the major part of the misactivated tyrosine is rejected as the result of a pretransfer correction consisting of the fast hydrolysis (k'h approximately equal to 20 s-1) of the enzyme-bound noncognate adenylate induced by the binding of native tRNAPhe. The transfer step itself is found to be non-specific, as the rate constant is almost the same for phenylalanine and tyrosine. This result is supported by the observation that tyrosine and phenylalanine are also transferred at the same rate to tRNAPheox-red. It is shown that the integrity of the 3'-terminal adenosine of the tRNA is critical for triggering the pretransfer hydrolysis of enzyme-bound noncognate aminoacyl adenylate. A detailed kinetic analysis is presented that shows that the observed rate constant of tRNAPhe tyrosylation and the rate of disappearance of enzyme-tyrosyl adenylate complex are in fact apparent rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate depletion analysis as an approach to the pre-steady-state anticooperative kinetics of aminoacyl adenylate formation by tryptophanyl-tRNA synthetase from beef pancreas.

The formation of tryptophanyl adenylate catalyzed by tryptophanyl-tRNA synthetase from beef pancreas has been studied by stopped-flow analysis under conditions where the concentration of one of the substrates was largely decreasing during the time course of the reaction. Under such conditions a nonlinear regression analysis of the formation of the adenylate (adenylate vs. time curve) at several initial tryptophan and enzyme concentrations gave an accurate determination of both binding constants of this substrate. The use of the jackknife procedure according to Cornish - Bowden & Wong [ Cornish - Bowden , A., & Wong , J.J. (1978) Biochem. J. 175, 969-976] gave the limit of confidence of these constants. This approach confirmed that tryptophanyl-tRNA synthetase presents a kinetic anticooperativity toward tryptophan in the activation reaction that closely parallels the anticooperativity found for tryptophan binding at equilibrium. Both sites are simultaneously forming the adenylate. The dissociation constants obtained under the present pre-steady-state conditions for tryptophan are KT1 = 1.6 +/- 0.5 microM and KT2 = 18.5 +/- 3.0 microM at pH 8.0, 25 degrees C. The rate constant kf of adenylate formation is identical for both active sites (kf = 42 +/- 5 s-1). The substrate depletion method presently used, linked to the jackknife procedure, proves to be particularly suitable for the determination of the kinetic constants and for the discrimination between different possible kinetic models of dimeric enzyme with high substrate affinity. In such a case this method is more reliable than the conventional method using substrate concentrations in high excess over that of the enzyme.

Aminoacyl-nucleotide reactions: studies related to the origin of the genetic code and protein synthesis.

In the present paper, we report on the effect of pH and carbonate on the hydrolysis rate constants of N-blocked and free aminoacyl adenylate anhydrides. Whereas the hydrolysis of free aminoacyl adenylates seems principally catalyzed by OH-, the hydrolysis of the N-blocked species is also catalyzed by H+, giving this compound a U-shaped hydrolysis vs. pH curve. Furthermore, at pH's less than 8, carbonate has an extreme catalytic effect on the hydrolysis of free aminoacyl-AMP anhydride, but essentially no effect on the hydrolysis of N-blocked aminoacyl-AMP anhydride. Furthermore, the N-blocked aminoacyl-AMP anhydride is a very efficient generator of peptides using free glycine as acceptor. The possible significance of the observations to prebiological peptide synthesis is discussed.

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

The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses of adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A), 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+ ions, catalyzes the synthesis of ADP from three different substrates which all lead to enzyme-bound glycyl adenylate, that is, ATP, adenosine 5'-[beta, gamma-methylene]triphosphate and Ap4A. It was furthermore demonstrated that the only pathway by which a synthetase-catalyzed degradation of Ap4A can occur is through the reaction between inorganic phosphate and the enzyme-bound glycyl adenylate, synthesized from Ap4A. 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 gamma-phosphate group from ATP to nucleoside 5'-diphosphates. The importance of the observed reaction between inorganic phosphate and enzyme-bound aminoacyl adenylate in relation to the remaining catalytic activities of aminoacyl-tRNA synthetases is discussed, as well as the biological significance of the reaction.

Highly efficient peptide formation from N-acetylaminoacyl-AMP anhydride and free amino acid

We report here on the kinetics of formation of the N-blocked dipeptide, N-acetylglycylglycine (Acgly-gly), from N-acetylglycyl adenylate anhydride (Acgly-AMP) and glycine in aqueous solution at 25°C, and at various pH's. The reaction is of interest in that over a physiologically relevant pH range (6–8), peptide synthesis proceeds more rapidly than hydrolysis, even at those pH's at which this compound becomes increasingly susceptible to base-catalyzed hydrolysis. Under similar conditions, the corresponding unblocked aminoacyl adenylate anhydrides are considerably more unstable, and undergo appreciable hydrolysis in the presence of free amino acid. Because N-blocked aminoacyl adenylate anhydrides serve as model compounds of peptidyl adenylate anhydrides, these results suggest that primitive amino acid polymerization systems may have operated by cyclic reactivation of the peptidyl carboxyl group, rather than that of the incoming amino acid.

No arginyl adenylate is detectable as an intermediate in the aminoacylation of tRNAArg.

On the supposition that aminoacyl adenylate is a necessary intermediate in the reactions catalyzed by aminoacyl-tRNA synthetases, six possible reactions requiring this intermediate were tested. With arginyl tRNA synthetase from brewer's yeast they were all negative and with phenylalanyl-tRNA synthetase they were all positive. Therefore, no evidence for the formation of arginyl adenylate could be provided. This is in contrast to results published elsewhere. It was shown that the reaction proceeds through a quaternary complex. The aminoacylation of the tRNA is followed by a rearrangement of the quaternary complex that also affects the structure of the arginyl-tRNA.

Zinc(II)-dependent synthesis of diadenosine 5', 5"' -P(1) ,P(4) -tetraphosphate by Escherichia coli and yeast phenylalanyl transfer ribonucleic acid synthetases.

A new activity of Escherichia coli and yeast phenylalanyl-tRNA synthetases, the conversion adenosine 5' -triphosphate into diadenosine 5' ,5"' -P(1) ,P(4) -tetraphosphate, is reported. This activity is followed by (31)P NMR and chromatography on poly(ethylenimine)-cellulose. It is revealed by the addition of ZnCl2 to a reaction mixture containing the enzyme, ATP-Mg(2+), L-phenylalanine, and pyrophosphatase It reflects the reaction enzyme-bound phenylalanyl adenylate with ATP instead of PPi and strongly depends on the hydrolysis of pyrophosphate in the assay medium. The zinc dependence of this reaction parallels that of the inhibition of tRNA(phe) aminoacylation which is described in the accompanying paper [Mayaux, J. F., & Blanquet, S. (1981) Biochemistry (preceding paper in this issue)]. In the presence of an unlimiting pyrophosphatase activity, diadenosine tetraphosphate synthesis by E. coli and yeast phenylalanyl-tRNA synthetases occurs at maximal rates of 0.5 and 2 s-1, respectively (37 degrees C, pH 7.8, 150 mM KC1, 5 mM ATP, 10 mM MgCl2, 2 mM L-phenylalanine, and 80 muM ZnCl2). Under identical experimental conditions, E coli isoleucyl-, methionyl-, and tyrosyl-tRNA synthetases produce small amounts of diadenosine tetraphosphate at rates 2 or 3 orders of magnitude lower than that achieved by phenylalanyl-tRNA synthetase. In the case of E. coli phenylalanyl-tRNA synthetase, it is shown that the diadenosine tetraphosphate synthetase activity is accompanied by a diadenosinetetraphosphatase activity. This activity, actually supported by phenylalanyl-tRNA synthetase, is responsible for the appearance of ADP in the assay medium. It requires also the presence of both ZnCl2 and L-phenylalanine. The formation of ADP from diadenosine tetraphosphate and its reaction with enzyme-bound aminoacyl adenylate account for the appearance in the reaction mixture of diadenosine 5' ,5"' -P(1) ,P(3)-triphosphate, after that of diadenosine tetraphosphate. The significance of these findings in the context of the role of diadenosine tetraphosphate in controlling cellular growth is discussed.

Stoichiometry and composition of an aminoacyl-tRNA synthetase complex from rat liver.

The particulate aminoacyl-tRNA synthetases of rat liver were copurified about 1000-fold with more than 20% yields for individual synthetase activities. Measurements of aminoacylation activities showed that lysyl-, arginyl-, leucyl-, isoleucyl-, and methionyl-tRNA synthetases in the purified complex cosedimented at 18 S. The molecular weight of the synthetase complex is about one million, as estimated by gel filtration. The stoichiometry of the synthetase in the complex was determined by active site titration with aminoacyl adenylates. Results indicate that the 18S synthetase complex contains one subunit of methionyl-tRNA synthetase and two subunits of lysyl-tRNA synthetase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the 18S synthetase complex contains eight major protein bands. Proteins with subunit molecular weights of 104,000, 92,000, 69,000, and 67,000 are present in molar ratios of 1:1:2:2, while proteins with subunit molecular weights of amounts. These results suggest that the particulate aminoacyl-tRNA synthetases exist as a heterotypic multienzyme complex with defined structure.

Stimulation of the transfer reaction of aminoacyl-tRNA synthetases by cations

The transfer reaction of amino acid from aminoacyl adenylate to tRNA catalyzed by some aminocyl-tRNA synthetases from Escherichia coli (isoleucine and lysine) is activated by a variety of cations, exemplified by Mg 2+, spermine, and NH 4 +. The phenylalanylt-RNA synthetase of E. coli has an absolute requirement for a divalent cation, Mg 2+ or Mn 2+, and is not activated by amines. The arginine enzyme is fully active without added cations.

Alternative pathways for editing non-cognate amino acids by aminoacyl-tRNA synthetases.

Evidence is presented that the editing mechanisms of aminoacyl-tRNA synthetase operate by two alternative pathways: pre-transfer, by hydrolysis of the non-cognate aminoacyl adenylate; post-transfer, by hydrolysis of the mischarged tRNA. The methionyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus and isoleucyl-tRNA synthetase from E. coli, for example, are shown to reject misactivated homocysteine rapidly by the pre-transfer route. A novel feature of this reaction is that homocysteine thiolactone is formed by the facile cyclisation of the homocysteinyl adenylate. Valyl-tRNA synthetases, on the other hand, reject the more readily activated non-cognate amino acids by primarily the post-transfer route. The features governing the choice of pathway are discussed.

Purification and properties of alanine tRNA synthetase from Escherichia coli A tetramer of identical subunits.

Escherichia coli alanine tRNA synthetase has been purified from a strain which carries the gene on a recombinant pBR322 plasmid. Several per cent of the soluble protein can be obtained as alanine tRNA synthetase in the plasmid containing host cell. The enzyme was proven to be an alpha 4 tetramer with a Mr = 380,000 by the following criteria: i) a single band of Mr = 95,000 was found in sodium dodecyl sulfate gel electrophoresis; ii) gel filtration chromatography gives a single peak at a Mr = 360,000 for the native enzyme; iii) dimethyl suberimidate cross-linking indicates a tetrameric structure; iv) a single undecapeptide N-terminal sequence was found which is Ser-Lys-Ser-Thr-Ala-Glu-Ile-Arg-Gln-Ala-Phe. Limited proteolysis generates a fragment of the native enzyme. The fragment has a Mr = 48,000 (one half that of the native subunit) and, in contrast to the native enzyme, oligomerization of the fragment could be not detected either by gel filtration chromatography or by dimethyl suberimidate cross-linking. The fragment is derived from the NH2-terminal half of the native subunit as shown by their common decapeptide NH2-terminal amino acid sequences. The ATP-PPi exchange activity of 1 mol of fragment is identical with that of 1 mol of native subunit, but aminoacylation activity is absent from the fragment. Therefore, in the fragment, the aminoacyl adenylate formation activity is cleanly separated from tRNA aminoacylation and subunit association properties. These results also mean that, with respect to aminoacyl adenylate formation activity, each subunit in the native tetramer acts independently.

Valyl-tRNA synthetase form yellow lupin seeds: hydrolysis of the enzyme-bound noncognate aminoacyl adenylate as a possible mechanism of increasing specificity of the aminoacyl-tRNA synthetase.

Valyl-tRNA synthetase form yellow lupin seeds: hydrolysis of the enzyme-bound noncognate aminoacyl adenylate as a possible mechanism of increasing specificity of the aminoacyl-tRNA synthetase.

Synthesis of amino acyl adenylates using the tert-butoxycarbonyl protecting group.

Alayl, [ 14c]-alanyl, phenylalanyl, and methionyl adenylates have been synthesized in high yields and relatively good purities. Elemental analysis 1H nmr and ir spectra have been utilized for the characterization of these extremely labile compounds. The present synthesis, which uses readily available N-tert-butoxycarbonyl amino acids, is compared with previous methods.

Establishing the misacylation/deacylation of the tRNA pathway for the editing mechanism of prokaryotic and eukaryotic valyl-tRNA synthetases

The valyl-tRNA synthetases from Bacillus stearothermophilus and Escherichia coli have been shown to edit the mistaken activation of L-a-aminobutyrate (aBut) by first misacylating tRNAVa1 and then specifically deacylating the aBut-tRNAVa'. This pathway was established initially by rapid quenching and sampling experiments which detected the transiently formed aBut-tRNAVa' on the addition of tRNAVa' to the preformed and isolated EaBut -AMP complex. The misacylation/deacylation pathway of editing was then shown to occur in the normal reaction in the steady state by isolation of the mischarged tRNA. For example, [14C]-aB~t-tRNAVa' ( E . coli) can be isolated from a steady-state reaction mixture containing [14C]-aBut (0.9 mM), enzyme (71 pM), tRNAVa' (72 pM), and ATP (8 mM), a t the steady-state concentration predicted from the measured deacylation rate constant (50 s-') and the steady-state kinetic constants for the ATP/pyroT h e fidelity of protein biosynthesis is far higher than expected from simple kinetic and thermodynamic arguments. One link in the synthetic chain where errors are likely to occur is a t the ' From the MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England. Received June 13, 1978; reaised manuscript received December 1 I , 1978. *Address from Sept 1978 to Sept 1979: Department of Biochemistry, Stanford University Medical Center, Stanford, CA 94305. 0006-2960/79/0418-1238$01 .OO/O phosphatase activity under these conditions. The same pathway appears to operate for the rejection of threonine by the eukaryotic valyl-tRNA synthetase since, on mixing the enzyme-bound threonyl adenylate and tRNA from yeast, transiently formed Thr-tRNAVa' is observed and its deacylation rate constant estimated to be greater than 80 s-'. The rate-determining step of the valyl-tRNA synthetase catalyzed deacylation of aBut-tRNAVa' is shown to be different in nature as well as in magnitude from that of Val-tRNAVa'. The rate of the editing reaction is independent of pH between 6 and 7.8 and is unaffected by the addition of aminoacyl adenylate, whilst the rate of deacylation of Val-tRNAva' follows an ionization of pK, = 8 and is decreased by the addition of aminoacyl adenylate. A unified scheme is presented that could account for the specificity in the deacylation reaction. aminoacylation of tRNA, for it is here that the cell has to distinguish between amino acids that are structurally very similar. The discrimination is accomplished by: (a) the aminoacyl-tRNA synthetases discriminating as precisely as possible between competing amino acids by the preferential binding of the cognate amino acid; (b) where necessary, the enzymes having evalved an editing function whereby mistakenly aminoacylated intermediates or products are hy

Phosphonate analogues of aminoacyl adenylates

Phosphonate analogues of aminoacyl adenylates

Yellow lupin ( Lupinus luteus) aminoacyl-tRNA synthetases : Isolation and some properties of enzyme-bound valyl adenylate and seryl adenylate

As a continuation of our studies on plant (yellow lupin, Lupinus luteus) aminoacyl-tRNA synthetases we describe here formation and some properties of valyl-tRNA synthetase-bound valyl adenylate (E Val(Val-AMP)) and seryl-tRNA synthetase-bound seryl adenylate (E Ser(Ser-AMP)). Valyl-tRNA synthetase-bound valyl adenylate was detected and isolated by several approaches in the pH range 6–10. In that range inorganic pyrophosphatase increases the amount of valyl adenylate by a factor 1.8 regardless of pH. 50% of valine from the E Val(Val-AMP) complex isolated by Sephadex G-100 gel filtration was transferred to tRNA with a rate constant greater than 4 min −1 (pH 6.2, 10°C). The ratio of valine to AMP in the enzyme-bound valyl adenylate is 1 : 1 and it is not changed by the presence of periodate-oxidized tRNA. In contrast to enzyme-bound valyl adenylate, formation of E Ser(Ser-AMP) is very sensitive to pH. Inorganic pyrophosphatase increases the amount of seryl adenylate by a factor 6 at pH 8.0 and 30 at pH 6.9. 60% of serine from the E Ser(Ser-AMP) complex was transferred to tRNA with a rate constant greater than 4 min −1 (pH 8.0, 0°C). The ratio of serine to AMP in the enzyme-bound seryl adenylate is 1 : 1. The rate of synthesis of the enzyme-bound aminoacyl adenylates was measured by ATP-PP i exchange. Michaelis constants for the substrates of valyl-tRNA and seryl-tRNA synthetases in ATP-PP i exchange were determined. Effects of pH, MgCl 2 and KCl on the initial velocity of aminoacyl adenylate formation are described. For comparison, catalytic indices in the aminoacylation reactions catalyzed by both lupin enzymes are given and effects of pH, MgCl 2 and KCl on tRNA aminoacylation are presented as well. Under some conditions, e.g. at low pH or high salt concentration, lupin valyl-tRNA and seryl-tRNA synthetase are active exclusively in ATP-PP i exchange reaction.

Phosphonate analogues of aminoacyl adenylates.

Phosphonomethyl analogues of glycyl phosphate and valyl phosphate, i.e. NH2-CHR-CO-CH2-PO(OH)2, were synthesized and esterified with adenosine to give analogues of aminoacyl adenylates. The interaction of these adenylate analogues with valyl-tRNA synthetase from Escherichia coli was studied by fluorescence titration. The analogue of valyl phosphate has an affinity for the enzyme comparable with that of valine, but that of valyl adenylate is bound much less tightly than either valyl adenylate or corresponding derivative of valinol. The affinity of the analogue of glycyl adenylate was too low to be measured. We conclude that this enzyme interacts specifically with both the side chain and the anhydride linkage of the adenylate intermediate.