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1.
In the biosynthesis of proteins, each amino acid passes from the aminoacyl adenylate to become an amino acid ester and finally a 2' (3') peptidyl ester of the AMP residue at the end of a tRNA. Consequently, the chemistry of protein synthesis is the chemistry of aminoacyl and peptidyl AMP. Our data has revealed properties of 5'-AMP and its esters which should allow the preferential catalytic synthesis of L-amino acid peptides via a bis(2', 3'-aminoacyl) ester intermediate. Results in this paper concern one step in the proposed process and show that preexisting Ac-L-Phe monoester reacts about 2.5-times faster to form diester than preexisting Ac-D-Phe monoester.  相似文献   

2.
Modified Tyr-tRNATyr and Phe-tRNAPhe species from yeast having the aminoacyl residue bound specifically to the 2' and 3' position of the terminal adenosine, respectively, were investigated for their ability to form ternary complexes with Escherichia coli elongation factor Tu and GTP. Both Tyr-tRNATyr-CpCpA (2'd) and Tyr-tRNATyr-CpCpA(3' d) derivatives which are esterified with the amino acid on the 3' and 2' position respectively and which lack the vicinal hydroxyl were able to form ternary complexes. The stability of these ternary complexes was lower than in the case of native Tyr-tRNATyr-CpCpA. Tyr-tRNATyr-CpCpA(3' d) having the amino acid attached to the 2' position interacted considerably more strongly with EF-Tu - GTP than Tyr-tRNATyr-CpCpA(2' d). Ternary complex formation was observed with neither Phe-tRNAPhe-CpCpA(2'NH2) nor Phe-tRNAPhe-CpCpA(3'NH2). It is concluded that 2' as well as 3' isomers of native aminoacyl-tRNA can be utilized for ternary complex formation but in a following step a uniform 2'-aminoacyl-tRNA - EF-Tu - GTP complex is formed. Although the free vicinal hydroxyl group of the terminal adenosine is not absolutely required, replacement of the ester linkage through with the amino acid is attached to tRNA by an amide linkage leads to loss of ability to interact with elongation factor Tu.  相似文献   

3.
M Taiji  S Yokoyama  T Miyazawa 《Biochemistry》1983,22(13):3220-3225
The rates of migration of the aminoacyl group (transacylation) between 2'-O-(aminoacyl)-tRNA and 3'-O-(aminoacyl)-tRNA were studied by the nuclear magnetic resonance (NMR) analyses of 3'-terminal fragment models, with regard to the significance of transacylation in the process of protein biosynthesis. 2'(3')-O-L-Alanyladenosine, -valyladenosine, -isoleucyladenosine, -phenylalanyladenosine, and -methionyladenosine, and 2'(3')-O-L-phenylalanyladenosine 5'-phosphate and methionyladenosine 5'-phosphate were chemically synthesized, and the rates of transacylation in deuterated buffer were directly measured by the NMR saturation transfer method. The dependences of transacylation rates on p2H and temperature were analyzed. The results indicate that the transacylation rates are significantly affected by the ionization states of the alpha-amino group of the amino acid moiety but not by the presence of the 5'-phosphate group of the adenylate moiety. The second-order rate constants for the base-catalyzed transacylation reactions were also determined for the ionized form (with alpha-N2H3+ group) of (aminoacyl)adenosines. The transacylation rates of (aminoacyl)adenosines in 1H2O solution at p1H 7.3 and 37 degrees C (intracellular environment) were evaluated as 3-11 s-1 for the 2' leads to 3' transacylation and 1-4 s-1 for the 3' leads to 2' transacylation, indicating that the transacylation rate of free aminoacyl-tRNA is slower than the overall rate of polypeptide chain elongation per ribosome. This suggests the presence of some enzymatic factor for enhancing the transacylation rates of aminoacyl-tRNAs in the polypeptide chain elongation process in vivo.  相似文献   

4.
The chemical synthesis of 2'(3')-O-L-phenylalanyl derivatives of nebularine (Ld), 6-methoxynebularine (Ih), N6,N6-dimethyladenosine (Lk), 6-methylthionebularine (Lo), 8-bromoadenosine (Lr), tubercidin (Iu), and 3'-O-L-phenylalanyl derivatives of 1-(beta-D-arabinofuranosyl)cytosine (IIIg) and 9-(beta-D-arabinofuranosyl)adenine (IIIl) is described. 2'(3')-O-(3-Phenyl)propionyladenosine (Iv) was obtained by reaction of adenosine with ethyl 3-phenylorthopropionate and subsequent hydrolysis of the orthoester intermediate IV with formic acid. Compounds Id, Ih, Ik, Io, and Iu were active in the release of Ac-Phe from N-Ac-Phe-tRNA-poly(U)-70S ribosome complex: at 0.01 mM the release of Ac-Phe was 50-100% of that of A-Phe. At 1 mM, compounds Ir and IIIg released 30 and 25% of Ac-Phe relative to A-Phe whereas derivatives Iv and IIIl were virtually inactive. The results indicate the following conclusions regarding ribosomal peptidyltransferase activity of 2'(3')-O-aminoacyl nucleosides: (a) the presence of the 2'-hydroxy group in the ribo configuration is more important for a highly active substrate (A-Phe) than for one of moderate activity (C-Phe); (b) the heterocyclic (purine) residue is in the anti conformation although this requirement is not absolute; (c) the presence of the amino group of the aminoacyl moiety is required; (d) acceptor activity is dependent upon the substituent in the position 6 of the purine moiety.  相似文献   

5.
Guth E  Connolly SH  Bovee M  Francklyn CS 《Biochemistry》2005,44(10):3785-3794
Aminoacyl-tRNA synthetases (aaRS) join amino acids to their cognate transfer RNAs, establishing an essential coding relationship in translation. To investigate the mechanism of aminoacyl transfer in class II Escherichia coli histidyl-tRNA synthetase (HisRS), we devised a rapid quench assay. Under single turnover conditions with limiting tRNA, aminoacyl transfer proceeds at 18.8 s(-)(1), whereas in the steady state, the overall rate of aminoacylation is limited by amino acid activation to a rate of 3 s(-)(1). In vivo, this mechanism may serve to allow the size of amino acid pools and energy charge to control the rate of aminoacylation and thus protein synthesis. Aminoacyl transfer experiments using HisRS active site mutants and phosphorothioate-substituted adenylate showed that substitution of the nonbridging Sp oxygen of the adenylate decreased the transfer rate at least 10 000-fold, providing direct experimental evidence for the role of this group as a general base for the reaction. Other kinetic experiments revealed that the rate of aminoacyl transfer is independent of the interaction between the carboxyamide group of Gln127 and the alpha-carboxylate carbon, arguing against the formation of a tetrahedral intermediate during the aminoacyl transfer. These experiments support a substrate-assisted concerted mechanism for HisRS, a feature that may generalize to other aaRS, as well as the peptidyl transferase center.  相似文献   

6.
In order to elucidate the substrate specificity of alanyl-tRNA synthetase, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (Ala-SA), an analogue of alanyl-AMP, was chemically synthesized. Its binding ability is similar to that of the substrate based on the inhibitory activity for the aminoacylation of alanyl-tRNA synthetase. Taking advantage of the stable sulfamoyl bond of Ala-Sa, compared with the highly labile aminoacyl bond of alanyl-AMP, the molecular conformation of the former inhibitor was studied by X-ray single crystal analysis. Crystal data are as follows: C13H19N7O7S.2H2O, space group C2, a = 39.620(6), b = 5.757(1), c = 20.040(3) A, beta = 117.2(1) degrees, V = 4065(9) A3, Z = 8, and final R = 0.065 for 2785 independent reflections of F(2)0 greater than or equal to 2 sigma (F0)2. In the crystal, the molecule is in a zwitterionic state with the terminal amino group protonated and sulfamoyl group deprotonated, and takes an open conformation, where the L-alanine moiety is located far from the adenosine moiety with gauche/trans and trans orientations about the exocyclic C(4')-C(5') and C(5')-O(5') bonds, respectively. The conformation of the adenosine moiety is anti for the glycosyl bond and C(3')-endo for the ribose puckering, and alanine is in the usually observed trans region for the psi torsion angle. The molecular dimensions of the sulfamoyl group are nearly the same as those of the phosphate group. The biological significance of the observed Ala-SA conformation is discussed in relation with the molecular conformation of tyrosyl-AMP complexed with tyrosyl-tRNA synthetase.  相似文献   

7.
In response to nutritional stress conditions, Bacillus brevis produces the cyclodecapeptide antibiotic tyrocidine via tyrocidine synthetase, a multifunctional non-ribosomal peptide synthetase. The apo-form of tyrocidine synthetase 1 forms adenosine (5')tetraphospho(5')adenosine, when incubated with MgATP(2-), amino acid and inorganic pyrophosphatase. The synthesis is an intrinsic property of the adenylation domain, is strictly dependent upon the amino acid, and proceeds from a reverse reaction of adenylate formation involving a second ATP molecule. In the presence of tri- or tetrapolyphosphate preferential synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate occurs, respectively. A potential involvement of adenosine (5')-n-phospho(5')adenosine in the regulation of the biosynthetic process has been suggested.  相似文献   

8.
The detailed synthesis of (bis)aminoacyl-pdCpAs and the corresponding singly and tandemly activated tRNAs is reported. The synthetic pathway leading to these compounds has been validated for simple amino acid residues as well as for amino acids bearing more complex side chains. Protection/deprotection strategies are described. For the bisaminoacylated tRNAs, both the synthesis of tRNAs bearing the same amino acid residue at the 2' and 3' positions and tRNAs bearing two different aminoacyl moieties are reported. Further, it is shown that the tandemly activated tRNAs are able to participate in protein synthesis.  相似文献   

9.
The catalytic competence of gramicidin S synthetase 2 (GS2) was determined by following the kinetics of PP(i) generation using active site titration measurements with [gamma-(32)P]ATP. The initial 'burst' of product formation can be correlated to the generation of the aminoacyl adenylate:enzyme complexes at the four amino acid activation domains and the subsequent aminoacylation of carrier domains, followed by a slow linear turnover of substrate due to breakdown of the intermediate. Simultaneous activation of all four amino acid substrates at a saturating concentration displayed a consumption of 8.3 ATP/GS2. In the presence of single amino acids, a binding stoichiometry higher than the anticipated two ATP per active site was obtained, implying misactivation at non-cognate domains. Breakdown of acyladenylate intermediates reflects a possible corrective mechanism by which the enzyme controls the fidelity of product formation.  相似文献   

10.
Reasons for believing that primitive mechanisms of translation may have employed thiol esters of the amino acids rather than oxygen esters are summarized. It is suggested that coenzyme A (HSCoA), which fulfills the role of aminoacyl transfer in the synthesis of peptide antibiotics, is a primitive analogue of tRNA which performs a similar role in protein synthesis. HSCoA—an adenylic acid moiety containing phosphates esterified at the 3′ and 5′ positions and linked to a peptide-like structure terminating in a reactive thiol—possesses chemical features suggestive of both peptides and polynucleotides. Examination of the chemistry of HSCoA-like molecules shows that a rather similar compound can carry out a repeating intramolecular peptide synthesis in the absence of enzymes. Condensation of further nucleotides onto the adenylic acid moiety gives rise to parallel modes of peptide and oligonucleotide synthesis. A “self-improving” ability to select available amino acids is inherent in the proposed mechanism of peptide synthesis. The hypothesis plausibly explains the universal occurrence of a sulphur-containing amino acid at the N terminus of nascent proteins.  相似文献   

11.
The reactivity order of O-deisopropylidenation of the three isopropylidene protecting groups of 2',6'-di-O-benzyl-2,3:5,6:3',4'-tri-O-isopropylidenelactose dimethyl acetal (2) with various reagents was established. The 5,6-acetal group was, although to a limited extent, more reactive as compared with the 3',4' group, while the 2,3-O-isopropylidene group was definitely less reactive. Conditions were determined for the direct preparation of the 5,6,3',4'-tetraol 5 (60% aqueous acetic acid, room temperature, 48 h, 73% yield) and the 5,6-diol 4 (propylene glycol and p-toluenesulphonic acid in dichloromethane, 46% yield). The diacetonated derivative 3, formally arising from a selective 3',4'-O-deisopropylidenation, was obtained in high yield (90%) through a selective acetonation with 2-methoxypropene of the tetraol 5.  相似文献   

12.
Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.  相似文献   

13.
C Scalfi-Happ  E Happ  S Ghag  S Chládek 《Biochemistry》1987,26(15):4682-4688
Specific syntheses of 2'(3')-O-aminoacyl oligoribonucleotides C-C-A-Gly (12), C-C-A(AcGly) (7), U-C-C-A-Gly (17), and C-U-C-C-A-Gly (19), which are the 3'-terminal sequences of Escherichia coli Gly-tRNA (or AcGly-tRNA, respectively) are described. Compounds 12, 17, and 19 were synthesized by employing the benzotriazolyl phosphotriester approach with the following protection groups on the components: benzoyl for the heterocyclic amino groups, 2-chlorophenyl group for internucleotide phosphate protection, dimethoxytrityl and levulinoyl groups for blocking of the 5'-hydroxyl, methoxytetrahydropyranyl group for protection of the 2'-hydroxyl functions, and N-(benzyloxycarbonyl)orthoglycinate as the masked aminoacyl group simultaneously protecting the 2',3'-cis diol group of the 3'-terminal adenosine moiety. The fully protected tri-, tetra-, and pentanucleotides were obtained via 5'-extension of di- and trinucleotide blocks after prior selective removal of the 5'-O-levulinoyl group with hydrazine. The blocked derivatives 11, 16, and 18 were totally deprotected by reactions with NH4OH, H+, and H2/Pd to yield the target compounds 12, 17, and 19 in good yields. C-C-A(AcGly) (7) was synthesized according to a stepwise procedure via activation of preformed diesters with (mesitylenesulfonyl)tetrazole. C-C-A-Gly (12), U-C-C-A-Gly (17), and C-U-C-C-A-Gly (19) were all acceptor substrates in the peptidyltransferase reaction with the Ac-Phe-tRNA-70S ribosome-poly(U) system. All three models also promoted EF-Tu-70S ribosome GTP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
The rate of transfer of amino acid from enzyme-bound aminoacyl adenylate to tRNA has been compared with the rate of esterification of free amino acid. The approach of L?vgren et al. (L?vgren, T. N. E., Heinonen, J., and Loftfield, R. B. (1975) J. Biol. Chem. 250, 3854-3860) was used, with 14C in the aminoacyl adenylate and 3H in the free amino acid and with both the lysine and isoleucine systems of Escherichia coli. In both systems kinetic analyses show more rapid transfer from the preformed enzyme complex when interference by the back reaction with inorganic pyrophosphate was eliminated. Parallel experiments, in which the amount of enzyme complex was measured, confirmed that aminoacyl adenylate is an intermediate in both systems. No evidence was found for an alternative mechanism.  相似文献   

15.
The cysteinyl transferase mycothiol ligase, or MshC, catalyzes the fourth step in the biosynthesis of the small molecular weight thiol mycothiol. MshC is essential for growth of Mycobacterium tuberculosis. Two groups of known aminoacyl tRNA synthetase inhibitors were evaluated for inhibition of M. tuberculosis MshC including aminoacyl adenosine analogs and natural products. Using enzyme assays, isothermal titration calorimetry and NMR, we show that MshC is selectively inhibited by cysteinyl sulfamoyl adenosine, and that discrimination occurs at the amino acid moiety.  相似文献   

16.
1. From NMR, IR and visible absorption studies of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), 2'(or 3')-O-(2, 4, 6-trinitrophenyl) adenosine (TNP-Ad(, and 1-(2'-hydroxyethoxy)-2, 4, 6-trinitrobenzene (TNP-EG), it was concluded that there is an intramolecular interaction between the base and 2, 4, 6-trinitrophenyl (TNP) moieties in the TNP-ATP molecule. 2. A broad new absorption band was observed in the 530-630 nm region when excess indole was added to reaction mixtures containing TNP-ATP dissolved in 50% methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, TNP-ATP and TNP-Ad underwent spectral shifts in the 400-550 nm region. The formation of a 1:1 complex apparently occurred between TNP-ATP and aromatic amino acid derivatives, and the complex with N-acetyltryptophan was stable in 50% methanol. The difference spectrum of TNP-EG vs. TNP-ATP closely resembled that induced by the addition of N-acetyltryptophan to the TNP-ATP solution. 3. The binding of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) to heavy meromyosin (HMM) was studied by the rapid gel equilibrium method using Sephadex G-25. A dissociation constant of 1.4 muM and a maximum binding number of 1.8 were obtained in 0.15 M KCl, 10 mM MgCl2, and 50 mM Tris-HCl (pH 8.0) at 25 degrees. TNP-ADP bound to the enzyme caused a characteristic spectral shift in the visible region. This spectral shift was explained in terms of an interaction between tryptophanyl residues and the adenine base of TNP-ADP bound to the enzyme. TNP-ADP quenched the tryptophanyl fluorescence, but TNP-EG and TNP-Ad did not. In the presence of 6 M guanidine hydrochloride, TNP-ADP scarcely quenched the tryptophanyl fluorescence, its effect being comparable to that of TNP-Ad.  相似文献   

17.
After deadenylation, most cytoplasmic mRNAs are decapped and digested by 5' to 3' exonucleases in Saccharomyces cerevisiae. Capped and deadenylated mRNAs are degraded to a lesser extent by 3' to 5' exonucleases. We have used a method, based on the electroporation of in vitro synthetised mRNAs, to study the relative importance of these two exonucleolytic pathways under stress conditions. We show that derepression of GCN4 upon amino acid starvation specifically limits the 5'-to-3'-degradation pathway. Because adenosine 3'-5' biphosphate (pAp), which is produced by Met16p, inhibits this degradation pathway to a comparable extent, we were prompted to analyse the role of Met16p in this phenomenon. We show that the inhibitory effects of amino acid limitation on 5' to 3' mRNA degradation are absent in a met16 mutant. We therefore conclude that the GCN4 dependence of MET16 expression is responsible for the decrease in 5' to 3' digestion under stress conditions and that cells use pAp as a signal to limit 5' to 3' RNA degradation under stress conditions. Because 3' to 5' mRNA degradation is unaffected, the relative importance of this pathway in the decay of certain RNAs may be increased under stress conditions.  相似文献   

18.
Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNAPro. The most important functional element of this catalytic mechanism is the 2′-OH group of the terminal adenosine 76 of Ala-tRNAPro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3′-O atom of A76.  相似文献   

19.
The peptide part of CoA, 4'-phosphopantetheine, has been identified as an essential cofactor in in the biosynthesis of fatty acids, polyketides, depsipeptides, peptides, and compounds derived from both carboxylic and amino acid precursors, like rapamycin. The cofactor is attached to a unique protein moiety, referred to as acyl carrier protein, aminoacyl carrier protein, or peptidyl carrier protein. These carrier proteins are either associated to enzyme complexes (type II) or integrated within multifunctional polypeptide chains (type I). The cofactor is added in a post-translational modification reaction by highly specific transferases, acting on CoA. The functions of carrier proteins in directed condensation reactions are: (1) the selection of substrates to be attached as thioesters, (2) the stabilization of intermediates, (3) the presentation of intermediates for modification by associated enzyme activities, (4) facilitation of the directed condensation reactions of two adjacent intermediates, and (5) assistance in the termination reaction(s) leading to product release.  相似文献   

20.
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