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W Freist

Publications and source records attributed to W Freist.

At least 37 records · Page 2Linked to original sources
PubMed1987

Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-A(3'NH2).

For discrimination between isoleucine and the other 19 naturally occurring amino acids by isoleucyl-tRNA synthetases from baker's yeast and from Escherichia coli MRE 600 discrimination factors have been determined from kcat and Km values in aminoacylation of the modified tRNA(Ile)-C-C-A(3'NH2). Discrimination factors D1 are products of an initial discrimination factor and a proof-reading factor: D1 = I1.II1. From discrimination factors and AMP formation stoichiometry factors I1 and II1 were calculated. D1 values obtained with the enzyme from E. coli are generally higher than those observed with the yeast enzyme, in some cases up to ten times. With both enzymes low D1 values are found for cysteine, valine, and tryptophan (20-200), the highest values for glycine, alanine, and serine (600-4000). I1 values calculated for the E. coli enzyme are slightly higher (4-145) than the factors observed with the yeast enzyme (1-85), proof-reading factors II1 of the E. coli enzyme are scattering about a mean value about 70, those of the yeast enzyme about a mean value about 50. Initial discrimination factors I1 are directly related to hydrophobic interaction forces between the substrates and the enzymes. Plots of Gibbs free energy differences calculated from these factors are linearly related to the accessible surface areas of the amino acids. A hypothetical model of the binding site can be given in which selection of amino acids is achieved by hydrophobic forces and removal of steric hindrance.

Acylation↗
PubMed1987

Isoleucyl-tRNA synthetase from Escherichia coli MRE 600: discrimination between isoleucine and valine with modulated accuracy.

Discrimination between isoleucine and valine is achieved with different accuracies by isoleucyl-tRNA synthetase from E. coli MRE 600. The recognition process consists of two initial discrimination steps and a pretransfer and a posttransfer proofreading event. The overall discrimination factors D were determined from kcat and Km values observed in aminoacylation of tRNA(Ile)-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNA(Ile)-C-C-A(3'NH2) initial discrimination factors I1 and pretransfer proofreading factors II1 were calculated. Factors I1 were computed from ATP consumption and D1, the overall discrimination in aminoacylation of the modified tRNA; factors II1 were calculated as quotient of AMP formation rates. Initial discrimination factors I2 and posttransfer proofreading factors II2 were determined from AMP formation rates observed in aminoacylation of tRNA(Ile)-C-C-A. The observed overall discrimination varies up to a factor of about four according to conditions. Under standard assay conditions 72,000, under optimal conditions 144,000 correct aminoacyl-tRNAs are produced per one error while 1.1 or 1.7 ATPs are consumed. A comparison with isoleucyl-tRNA synthetase from yeast shows that both enzymes act principally with the same recognition mechanism, but the enzyme from E. coli MRE 600 exhibits higher specificity and lower energy dissipation and does not show such high variation of accuracy as observed with the enzyme from yeast.

Adenosine Monophosphate↗
PubMed1986

Biochemical comparison of the Neurospora crassa wild type and the temperature-sensitive and leucine-auxotroph mutant leu-5. Purification of the cytoplasmic and mitochondrial leucyl-tRNA synthetases and comparison of the enzymatic activities and the degradation patterns.

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 degrees C) and mutant (grown at 28 degrees C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28 degrees C and transferred then to 37 degrees C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37 degrees C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish and difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 degrees C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of approximately 1:600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme.

Adenosine Triphosphate↗
PubMed1986

Assignment of conserved amino acid residues to the ATP site in the protein kinase domain of the receptor for epidermal growth factor.

The ATP substrate site in the epidermal growth factor (EGF) receptor was mapped by using a series of 26 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP. The enzyme seems to interact specifically with the beta-phosphate in an ion-pair bond with the N-6 amino group at the adenine in a hydrogen bond. With ribosyl-2-aminopurine triphosphate and GTP, the enzyme most likely recognizes the 2-amino group in a hydrogen bond. This high specificity for ATP and GTP is unique for the ATP site in the EGF receptor among all investigated protein kinases. The available data on the interaction between ATP derivatives and protein kinases were used to assign conserved amino acid residues found in diverse protein kinases to the ATP site in this type of enzyme.

Adenosine Triphosphate↗
PubMed1986

Isoleucyl-tRNA synthetase from baker's yeast. Influence of substrate concentrations on aminoacylation pathways, discrimination between tRNAIle and tRNAVal, and between isoleucine and valine.

The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast. For the cognate substrates isoleucine and tRNAIle two Km values each differing by a factor about five were determined; the higher values were observed at concentrations higher than 1 microM, the lower values below 1 microM isoleucine or tRNAIle, respectively. At substrate concentrations below 1 microM also kcat values of the isoleucylation reaction are lowered. With the noncognate substrates valine and tRNAVal such differences could not be detected. The substrate ATP did not show any change of its Km value as far as the reaction was measurable. Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNAIle as the third one; pyrophosphate is the first product to be released, isoleucyl-tRNA the second, and AMP the third one. In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine. From kcat and Km values and from AMP formation rates discrimination factors for discrimination between tRNAIleII and tRNAValI as well as between isoleucine and valine were determined. In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant. The two different Km values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism. Two hypothetical catalytic cycles are postulated.

Adenosine Monophosphate↗
PubMed1985

Isoleucyl-tRNA synthetase from bakers' yeast: multistep proofreading in discrimination between isoleucine and valine with modulated accuracy, a scheme for molecular recognition by energy dissipation.

For discrimination between isoleucine and valine by isoleucyl-tRNA synthetase from yeast, a multistep sequence is established. The initial discrimination of the substrates is followed by a pretransfer and a posttransfer hydrolytic proofreading process. The overall discrimination factor D was determined from kcat and Km values observed in aminoacylation of tRNAIle-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNAIle-C-C-3'dA and tRNAIle-C-C-A (3'NH2), the initial discrimination factor I (valid for the reversible substrate binding) and the proofreading factor P1 (valid for the aminoacyl adenylate formation) could be determined. Factor I was computed from ATP consumption and D1, the overall discrimination factor for this partial reaction which can be obtained from kinetic constants, and P1 was calculated from AMP formation rates. Proofreading factor P2 (valid for aminoacyl transfer reaction) was determined from AMP formation rates observed in aminoacylation of tRNAIle-C-C-A and tRNAIle-C-C-3'dA. From the initial discrimination factor I and the AMP formation rates, discrimination factor DAMP in aminoacylation of tRNAIle-C-C-A can be calculated. These values deviate by a factor II from factor D obtained by kinetics which may be due to the fact that for acylation of tRNAIle-C-C-A an initial discrimination factor I' = III is valid. The observed overall discrimination varies up to a factor of 16 according to conditions. Under optimal conditions, 38 000 correct aminoacyl-tRNAs are produced per 1 error while the energy of 5.5 ATPs is dissipated. With the determined energetic and molecular flows for the various steps of the enzymatic reaction, a coherent picture of this new type of "far away from equilibrium enzyme" emerges.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Isoleucyl-tRNA synthetase from bakers' yeast: variable discrimination between tRNAIle and tRNAVal and different pathways of cognate and noncognate aminoacylation under standard conditions, in the presence of pyrophosphatase, elongation factor Tu-GTP complex, and spermine.

Error rates in discrimination between cognate tRNAIle and noncognate tRNAVal in the aminoacylation reaction with isoleucine catalyzed by isoleucyl-tRNA synthetase from yeast have been investigated in three sets of experiments under different assay conditions. The overall discrimination factor was first determined by isoleucylation of tRNAVal/tRNAIle mixtures. In the second set of experiments, the number of AMP molecules formed per Ile-tRNA in the cognate and noncognate reactions was measured. The higher AMP formation in the noncognate aminoacylation is assigned to a proofreading reaction step. The calculated proofreading factors and an estimated initial discrimination factor yield overall discriminations that are consistent with those obtained from the first set of experiments. In the third series of studies, the orders of substrate addition and product release of cognate and noncognate isoleucylation reactions were investigated by initial rate kinetic methods. From kcat and Km values, the overall discrimination factors were calculated and showed again a good coincidence with those observed in the preceding sets of experiments. Besides under standard assay conditions, aminoacylation reactions were studied in the presence of pyrophosphatase or elongation factor Tu-GTP complex, under addition of both these proteins, in presence of these two additional proteins and spermine at high and low magnesium concentrations, and under special conditions that favor misacylations. Furthermore, isoleucylation of tRNAIle was tested at increased and decreased pH in the standard enzyme assay. Variation of the assay conditions results in changing discrimination factors, which differ by a factor of about 10. Substitution of tRNAIle by tRNAVal in the isoleucylation reaction causes changes in substrate addition and product release orders and thus of the whole catalytic cycle. For aminoacylation of tRNAIle, four different orders of substrate addition and product release appear: the sequential ordered ter-ter, the rapid equilibrium sequential random ter-ter, the random bi-uni uni-bi ping-pong, and a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment. tRNAVal is aminoacylated in rapid equilibrium random ter-ter order, in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment, and in two bi-uni uni-bi ping-pong mechanisms. It is assumed that the different assay conditions can be regarded as a stepwise approximation to physiological conditions and that considerable changes in error rates may be also possible in vivo up to 1 order of magnitude.

Adenosine Monophosphate↗
PubMed1984

ATP analog specificity of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phosphorylase kinase.

The ATP analog specificities of the homogeneous cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase have been compared by the ability of 27 analogs to compete with ATP in the protein kinase reaction. Although the data suggest general similarities between the ATP sites of the two homologous cyclic-nucleotide-dependent protein kinases, specific differences especially in the adenine binding pocket are indicated. These differences in affinity suggest potentially useful ATP analog inhibitors of each kinase. For example, apparent autophosphorylation of the purified regulatory subunit of the cAMP-dependent protein kinase is blocked by nebularin triphosphate, suggesting that the phosphorylation is catalyzed by trace contamination of cGMP-dependent protein kinase. Some of the ATP analogs have also been tested using phosphorylase b kinase in order to compare this enzyme with the cyclic-nucleotide-dependent enzymes. All three protein kinases have high specificity for the purine moiety of ATP, and lower specificity for the ribose or triphosphate. The similarity between the ATP site of phosphorylase b kinase to that of the cyclic-nucleotide-dependent protein kinases suggests that it is related to them. The ATP analog specificities of enzymes examined in this study are different from those reported for several unrelated ATP-utilizing enzymes.

Adenosine Triphosphate↗
PubMed1983

Isoleucyl-tRNA synthetase from Baker's yeast. Catalytic mechanism, 2',3'-specificity and fidelity in aminoacylation of tRNAIle with isoleucine and valine investigated with initial-rate kinetics using analogs of tRNA, ATP and amino acids.

The aminoacylation of three modified tRNAIle species with isoleucine and with valine by isoleucyl-tRNA synthetase has been investigated by initial rate kinetics. For aminoacylation of tRNAIle-C-C-3'dA with isoleucine, a bi-bi uni-uni ping-pong mechanism has been found by bisubstrate kinetics and inhibition by products and by 3'dATP; for aminoacylation with valine a bi-uni uni-bi ping-pong mechanism. For isoleucylation of tRNAIle-C-C-A(3'NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni-bi uni-uni-uni ping-pong mechanism; for valylation of this tRNA a bi-bi uni-uni ping-pong mechanism is observed by bisubstrate kinetics and product inhibition. tRNAIle-C-C-2'dA was aminoacylated under modified conditions with isoleucine in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P[NH]P as product analog and by isoleucinol. Aminoacylation with valine is achieved in a rapid-equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3'dATP and valinol. All six reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle-C-C-A. The Km values of the three substrates and the kcat values of the six reactions are given. For aminoacylation at the terminal 2'OH group of the tRNA differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine have been calculated which result in discrimination factors of 181 and 167. For aminoacylation at the terminal 3'-OH group a difference of only 4.43 kJ and a low discrimination factor of only 6 is observed. Thus maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2'-OH group and conclusions drawn from experiments with modified tRNAs concerning 2',3'-specificity have led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNAs. The stability of Ile-tRNAIle-C-C-2'dA and Val-tRNAIle-C-C-2'dA, the lesser stability of Val-tRNAVal-C-C-2'dA and the instability of Thr-tRNAVal-C-C-2'dA are consistent with postulations for a 'pre-transfer' proofreading step for isoleucyl-tRNA synthetase and a 'post-transfer' hydrolytic editing step for valyl-tRNA synthetase at the terminal 3'OH group of the tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Isoleucyl-tRNA synthetase from Escherichia coli MRE 600. Different pathways of the aminoacylation reaction depending on presence of pyrophosphatase, order of substrate addition in the pyrophosphate exchange, and substrate specificity with regard to ATP analogs.

The substrate specificity of isoleucyl-tRNA synthetase from Escherichia coli MRE 600 with regard to ATP analogs has been compared with the results obtained with isoleucyl-tRNA synthetase from yeast. The enzyme from E. coli is less specific, the two enzymes exhibit different topographies of their active centres. The order of substrate addition to isoleucyl-tRNA synthetase from E. coli MRE 600 has been investigated by bisubstrate kinetics, product inhibition and inhibition by substrate analogs. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. As found for isoleucyl-tRNA synthetase from yeast, the results of the pyrophosphate exchange studies indicate the possibility of formation of E . Ile-AMP . ATP complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, release of pyrophosphate and subsequent addition of a second molecule of ATP. For the aminoacylation in the absence of pyrophosphatase, a rapid-equilibrium random ter addition of the substrates is found whereas the enzyme from yeast exhibits a steady-state ordered ter-ter mechanism; in the presence of pyrophosphatase the mechanism is bi-uni uni-bi ping-pong similarly as observed for the yeast enzyme. A comparison of inhibition patterns obtained with N(6)-benzyladenosine 5'-triphosphate under different assay conditions (spermine or magnesium ions, addition of pyrophosphatase) indicates that even more than two pathways of the aminoacylation may exist. The catalytic cycles of the two mechanisms derived from the observed orders of substrate addition and product release include the same enzyme substrate complex (E . tRNA . Ile-AMP) for the aminoacyl transfer reaction. The kcat values, however, are considerably different: kcat of the sequential pathway is about 40% lower than kcat of the ping-pong mechanism.

Adenosine Triphosphate↗
PubMed1982

Arginyl-tRNA synthetase from Escherichia coli K12: specificity with regard to ATP analogs and their magnesium complexes.

Fifteen analogs of ATP have been tested in the ATP/PPi pyrophosphate exchange and the aminoacylation of arginyl-tRNA synthetase from E. coli K12. Six compounds are substrates in both reactions, whereas seven of the triphosphates were inhibitors for both reactions. The Km, V and Ki values have been determined. The enzyme is less specific against base modifications of the ATP molecule than arginyl tRNA synthetase from baker's yeast in the aminoacylation and is inhibited by more base-modified compounds in the ATP/PPi exchange. The enzyme accepts 3'-deoxy-ATP as substrate and is inhibited by 2'-methoxy-ATP, whereas the reversed observation is made for the yeast arginyl-tRNA synthetase. The stoichiometry and association constants of complexes formed by six ATP analogs (four substrates and two inhibitors) and magnesium ions were investigated; five analogs form 1:1 complexes, one analog (3'-deoxy-ATP) binds two magnesium ions. The enzyme must accept different complexes formed with one or two magnesium ions as substrate, which must be different in structure from complexes proposed in literature.

Adenosine Triphosphate↗
PubMed1982

Phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria, and from Escherichia coli: substrate specificity relationship with regard to ATP analogs.

Twelve structural analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria and E. coli. Three compounds are substrates for all five phenylalanyl-tRNA synthetase, three are completely inactive, while the other ATP analogs show differing properties with the different enzymes. Their Km, Ki and V values have been determined. The importance of the amino group in Position 6, the nitrogen in Position 7 and an unsubstituted Position 8 of the purine moiety as well as the supposed anti-conformation of the glycosidic bond and coordination of the magnesium cation to N-7 appear to be conserved through evolution. Bulky substituents on the 2' and 3' of the ribose moiety are generally not tolerated. Graduation of substrate properties of some analogs are similar for the intracellular heterotopic isoenzymes from yeast and hen liver.

Adenosine Triphosphate↗
PubMed1981

The ATP substrate site of a cyclic-nucleotide-independent protein kinase from porcine liver nuclei.

The ATP substrate site of a second messenger-independent protein kinase of the type NII from porcine liver nuclei was mapped using a series of 30 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP; they range from 4 microM to 1.5 mM. For a comparison with data previously reported for the catalytic subunit of cAMP-dependent protein kinase I from rabbit skeletal muscle, the Ki values were transformed into delta delta values. These values are related to the Ki value of unsubstituted ATP and indicate the decrease of affinity caused by the different substitutions. With both enzymes the major binding affinity is derived from the interaction of the adenine base. The contributions of the two ribosyl OH groups are marginal and the triphosphate moiety interacts most strongly with its beta-phosphoryl group. Between the two enzymes the most striking differences, however, were observed for the specificity of the nucleobase interaction. While an unmodified N-6 amino group is required in the case of the cAMP-dependent protein kinase, the nuclear enzyme seems to tolerate extensive modification at this position, such as the introduction of a keto group or a bulky benzyl residue. Obviously, the ATP site of the nuclear kinase has an open cleft next to the N-6 of the adenine and binding of the adenine occurs by hydrophobic interaction without the formation of hydrogen bonds to any of the adenine nitrogens.

Adenosine Triphosphate↗
PubMed1981

Isoleucyl-tRNA synthetase from Baker's yeast. Action of ATP analogs in pyrophosphate exchange and aminoacylation, two pathways of the aminoacylation depending on concentration of pyrophosphate.

The order of substrate addition to isoleucyl-tRNA synthetase from baker's yeast has been investigated by steady-state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncompetitively with respect to ATP, namely purineriboside (= nebularin), 3'-deoxy-adenosine (= cordycepin), 8-amino-adenosine and 8-azido-adenosine 5'-triphosphates. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. Additionally, bisubstrate kinetics and product inhibition studies were carried out. The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates. The results of the pyrophosphate exchange studies are consistent with formation of E . Ile-AMP . ATP . Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and isoleucine. For the aminoacylation in the absence of pyrophosphatase an ordered ter-ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi-uni uni-bi ping-pong. Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter-ter mechanism.

Adenosine Triphosphate↗
PubMed1981

Arginyl-tRNA synthetase from Baker's yeast. Order of substrate addition and action of ATP analogs in the aminoacylation reaction; influence of pyrophosphate on the catalytic mechanism.

The order of substrate addition to arginyl-tRNA synthetase from baker's yeast has been investigated by bisubstrate kinetics, product inhibition and inhibition by three different inhibiting ATP analogs, the 6-N-benzyl, 8-bromo and 3'-deoxy derivatives of ATP, each acting competitively with respect to one of the substrates. The kinetic patterns are consistent with a random ter-ter mechanism, an addition of the three substrates and release of the products in random order. The different inhibitors are bound to different enzyme . substrate complexes of the reaction sequence. Addition of inorganic pyrophosphatase changes the inhibition patterns and addition of methylenediphosphonate as pyrophosphate analog abolishes the effect of pyrophosphatase, showing that the concentration of pyrophosphate is determinant for the mechanism of catalysis.

Acylation↗
PubMed1981

Survey on substrate specificity with regard to ATP analogs of aminoacyl-tRNA synthetases from E. coli and from Baker's yeast. Correlation to synthetase families.

The substrate specificity of twenty aminoacyl-tRNA synthetases from E. coli and thirteen enzymes from baker's yeast with regard to eight ATP analogs is investigated for a comparison of the active-site topography. The enzymes are arranged in a scheme of possible "enzyme families" and compared to earlier schemes.

Adenosine Triphosphate↗
PubMed1980

Phenylalanyl-tRNA, lysyl-tRNA, isoleucyl-tRNA and arginyl-tRNA synthetases. Substrate specificity in the ATP/PPi exchange with regard to ATP analogs.

The analogs of ATP have been tested in the ATP/PPi exchange reaction of phenylalanyl-tRNA, lysyl-tRNA, isoleucyl-tRNA and arginyl-tRNA synthetases from baker's yeast. Three compounds are substrates for phenylalanyl-tRNA, seven for lysyl-tRNA, two for isoleucyl-tRNA and five for arginyl-tRNA synthetase. Their Km and V values have been determined. No analog was an inhibitor. (3'-dATP), 3'-Deoxyadenosine 5'-triphosphate which is an inhibitor of the four enzymes in the aminoacylation reaction, becomes a good substrate in the PPi exchange. Additionally lysyl-tRNA synthetase accepts two analogs with modifications at position 6 of the purine and three analogs modified at the ribose moiety as substrates in the PPi exchange, whereas these compounds are inactive or inhibitors in the aminoacylation reaction. In general the enzymes are less specific in the ATP/PPi exchange and the results indicate a more sophisticated proof of the nucleotide moiety upon aminoacylation. This could occur with the aminoacyladenylate intermediate as well as with any other intermediate.

Adenosine Triphosphate↗