Biomedical subjects

W Freist

Publications and source records attributed to W Freist.

At least 19 recordsLinked to original sources

PubMed1999

Histidyl-tRNA synthetase.

Histidyl-tRNA synthetase (HisRS) is responsible for the synthesis of histidyl-transfer RNA, which is essential for the incorporation of histidine into proteins. This amino acid has uniquely moderate basic properties and is an important group in many catalytic functions of enzymes. A compilation of currently known primary structures of HisRS shows that the subunits of these homo-dimeric enzymes consist of 420-550 amino acid residues. This represents a relatively short chain length among aminoacyl-tRNA synthetases (aaRS), whose peptide chain sizes range from about 300 to 1100 amino acid residues. The crystal structures of HisRS from two organisms and their complexes with histidine, histidyl-adenylate and histidinol with ATP have been solved. HisRS from Escherichia coli and Thermus thermophilus are very similar dimeric enzymes consisting of three domains: the N-terminal catalytic domain containing the six-stranded antiparallel beta-sheet and the three motifs characteristic of class II aaRS, a HisRS-specific helical domain inserted between motifs 2 and 3 that may contact the acceptor stem of the tRNA, and a C-terminal alpha/beta domain that may be involved in the recognition of the anticodon stem and loop of tRNA(His). The aminoacylation reaction follows the standard two-step mechanism. HisRS also belongs to the group of aaRS that can rapidly synthesize diadenosine tetraphosphate, a compound that is suspected to be involved in several regulatory mechanisms of cell metabolism. Many analogs of histidine have been tested for their properties as substrates or inhibitors of HisRS, leading to the elucidation of structure-activity relationships concerning configuration, importance of the carboxy and amino group, and the nature of the side chain. HisRS has been found to act as a particularly important antigen in autoimmune diseases such as rheumatic arthritis or myositis. Successful attempts have been made to identify epitopes responsible for the complexation with such auto-antibodies.

Adenosine Triphosphate↗

PubMed1998

ATP binding site of P2X channel proteins: structural similarities with class II aminoacyl-tRNA synthetases.

The extracellular loop of P2X channel proteins contains a sequence stretch (positions 170-330) that exhibits similarities with the catalytic domains of class II aminoacyl-tRNA synthetases as shown by secondary structure predictions and sequence alignments. The arrangement of several conserved cysteines (positions 110-170) shows similarities with metal binding regions of metallothioneins and zinc finger motifs. Thus, for the extracellular part of P2X channel proteins a metal binding domain and an antiparallel six-stranded beta-pleated sheet containing the ATP binding site are very probable. The putative channel forming H5 part (positions 320-340) shows similarities with the enzyme motif 1 responsible for aggregation of subunits to the holoenzyme.

Adenosine Triphosphate↗

PubMed1998

Accuracy of protein biosynthesis: quasi-species nature of proteins and possibility of error catastrophes.

Yeast aminoacyl-tRNA synthetases act in a multi-step process when recognizing their cognate amino acids; this identification event includes "physical" binding and "chemical" proof-reading steps. However, the various enzymes use these single steps at different degrees, and their specificities with regard to the 20 naturally occurring amino acids deviate considerably from each other. The characteristic discrimination factors D were determined for seven synthetases in vitro: the highest specificity with D values between 28,000 and > 500,000 were observed with tyrosyl-tRNA synthetase, the lowest values between 130 and 1700 for lysyl-tRNA synthetase. The tested class I enzymes are more specific than the investigated class II enzymes, and it may be put into discussion whether this observation can be generalized. Error rates in amino acid recognition differ not only between the individual aminoacyl-tRNA synthetases but also considerably for different amino acids sorted by the same enzyme. Strikingly, all investigated enzymes exhibit a poor specificity in discrimination of cysteine and tryptophan from their cognate substrates, and these cases may be regarded as "specificity holes". In view of the observed specificities a protein consisting of 700 amino acids would contain maximally up to five "incorrect" residues, if the in vitro error rates are also valid under in vivo conditions. Therefore the terminus "quasi-species", an expression which was originally created for nucleic acids, is justified. The "quasi-species" nature of proteins may become important when genes are translated in different organisms with different accuracies of the translation apparatus. In such cases different "quasi-species" will be obtained. Using our data in mathematical models which predict the stability of protein synthesizing systems, we find that they are consistent with a stable yeast organism which is not prone to die by an "error catastrophe". However, this appears only if average values from our experiments are used for calculations. If a single compound, e.g. the arginine analog canavanine, is discriminated very poorly from the cognate substrate, or if the "specificity holes" get larger, an "error catastrophe" must be envisaged.

Amino Acids↗

PubMed1997

Glutaminyl-tRNA synthetase.

Among the twenty aminoacyl-tRNA synthetases glutaminyl-tRNA synthetase occupies a special position: it is one of only two enzymes of this family which is not found in all organisms, being mainly absent from gram positive eubacteria, archaebacteria and organelles. The E. coli GlnRS is relatively small with 553 amino acids and a molecular mass of 64.4 kDa and functions as a monomer. The mammalian enzymes are somewhat larger and can be parts of multienzyme complexes. Crystal structures were solved of E. coli GlnRS complexed with tRNA(Gln) and ATP, of this complex containing tRNA(Gln) replaced by unmodified tRNA(Gln), and of three complexes with mutated GlnRS enzymes. The GlnRS molecule consists of four domains, the catalytic site is located in the Rossman fold, typical for class I synthetases, and the reaction mechanism follows the normal adenylate pathway. The enzyme shows many similarities with glutamyl-tRNA synthetase; a common ancestor of both molecules is well established. In the E. coli system recognition of the cognate tRNA has been studied in many details using both natural and artificial mutants of tRNA(Gln) and of the enzyme: GlnRS recognizes mainly conventional parts of the tRNA molecule, namely some bases of the anticodon loop and parts of the acceptor stem.

Adenosine Triphosphate↗

PubMed1997

Glutamyl-tRNA sythetase.

Glutamyl-tRNA synthetase (GluRS) belongs to the class I aminoacyl-tRNA synthetases and shows several similarities with glutaminyl-tRNA synthetase concerning structure and catalytic properties. Phylogenetic studies suggested that both diverged from an ancestral glutamyl-tRNA synthetase responsible for the gluta-mylation of tRNA(Glu) and tRNA(Gln), and whose Glu-tRNA(Gln) product is transformed into Gln-tRNA(Gln) by a specific amidotransferase. This pathway is present in gram-positive and some gram-negative eubacteria, in some archae and in organelles, and was never found jointly with a glutaminyl-tRNA synthetase. Other gram-negative eubacteria and the cytoplasm of eukaryotes contain a glutamyl-tRNA synthetase specific for tRNA(Glu), and a glutaminyl-tRNA synthetase. Bacterial glutamyl-tRNA synthetases consist of about 500 amino acid residues, possess molecular masses of about 50 kDa, and act as monomers. In higher eukaryotes chimeric glutamyl-prolyl-tRNA synthetases were found, in a high molecular mass complex containing several other aminoacyl-tRNA synthetases. To date one crystal structure of a glutamyl-tRNA synthetase (Thermus thermophilus) has been solved. The molecule has the form of a bent cylinder and consists of four domains. The N-terminal half (domains 1 and 2) contains the 'Rossman fold' typical for class I synthetases and resembles the corresponding part of E. coli GlnRS, whereas the C-terminal half exhibits a GluRS-specific structure. As found for the other aminoacyl-tRNA synthetases the catalytic pathway of GluRS includes the formation of an aminoacyl adenylate in the first reaction step, but GluRS shares a special property with GlnRS and ArgRS: the ATP/PPi pyrophosphate exchange reaction is only catalyzed in the presence of the cognate tRNA. Compared with other aminoacyl-tRNA synthetases a relatively high number of investigations deals with recognition of tRNA(Glu) by GluRS. Besides interactions between the enzyme and the acceptor stem and the anticodon of tRNA(Glu), checking of the dihydrouridine arm and of the variable loop by GluRS are documented.

Acylation↗

PubMed1996

Phenylalanyl-tRNA synthetase from yeast and its discrimination of 19 amino acids in aminoacylation of tRNA(Phe)-C-C-A and tRNA(Phe)-C-C-A(3'NH2).

For discrimination between phenylalanine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for phenylalanine, discrimination factors, D, of 190-6300 have been determined from kcal and K(m) values. Generally, phenylalanyl-tRNA synthetase is more specific than the class II enzymes specific for Lys and Thr, but works with lower accuracy than the class I enzymes specific for IIe, Tyr, and Arg. In aminoacylation of tRNA(Phe)-C-C-A(3'NH2) discrimination factors D1 vary between 80-1610. Pre-transfer proof-reading factors II1 are in the range 2.3-74, post-transfer proof-reading factors II2 in the range 1.0-4.6, showing that pre-transfer proof-reading is the main correction step, post-transfer proofreading is less effective or negligible. Initial discrimination factors (I1 and I2) caused by differences in Gibbs free energies of binding between phenylalanine and non-cognate amino acids have been calculated assuming a two-step binding process. Factors I1 can be related to hydrophobic-interaction forces depending on accessible surface areas of the amino acids, factors I2 scatter about a low mean value and do not show any relation to amino acid structures or surfaces, indicating less checking of amino acid side chains in the putative second binding step.

Acylation↗

PubMed1996

Glycyl-tRNA synthetase.

Glycyl-tRNA synthetase, a class II aminoacyl-tRNA synthetase, catalyzes the synthesis of glycyl-tRNA, which is required to insert glycine into proteins. In a side reaction the enzyme also synthesizes dinuceloside polyphosphates, which probably participate in regulation of cell functions. Glycine is the smallest amino acid occurring in natural proteins, probably established as a protein component very early in evolution. Besides the amino and the carboxyl groups there is no functional group in the molecule. Alanine, the amino acid which is structurally most similar to glycine, possesses an additional methyl group as 'side chain'. Glycyl-tRNA synthetase is one of the few synthetases which exhibit different oligomeric structures in different organisms (alpha 2 beta 2 and alpha 2). The alpha 2 beta 2 enzymes exhibit similarities to PheRS (also an alpha 2 beta 2 enzyme). The alpha 2 forms belong to the subclass IIa enzymes with regard to sequence homologies. In eukaryotes the polypeptide is weakly associated with multienzyme complexes consisting of aminoacyl-tRNA synthetases. In the aminoacylation reaction a 'half-of-the-sites' mechanism as found for GlyRS from Bombyx mori is probably used by all glycyl-tRNA synthetases under in vivo conditions. Essentially, tRNAGly is recognized by GlyRS through standard identity elements in the anticodon region and in the acceptor stem. The last three facts may indicate that GlyRS is an enzyme which still possesses properties of a primordial aminoacyl-tRNA synthetase. Nine genes of glycyl-tRNA synthetases from six organisms have been sequenced. They encode synthetase subunits of chain lengths ranging from 300-700 amino acids. One crystal structure, that of the alpha 2 enzyme from Thermus thermophilus, has also been determined. The two subunits each possess three domains: the active site resembling that of aspartyl and seryl enzymes, a C-terminal anticodon recognition domain, and one domain which almost certainly interacts with the acceptor stem of tRNAGly. Antibodies against glycyl-RNA synthetase occur in the sera of patients suffering from polymyositis and interstitial lung disease.

Animals↗

PubMed1995

Lysyl-tRNA synthetase.

Lysyl-tRNA synthetase catalyses the formation of lysyl-transfer RNA, Lys-tRNA(Lys), which then is ready to insert lysine into proteins. Lysine is important for proteins since it is one of only two proteinogenic amino acids carrying an alkaline functional group. Seven genes of lysyl-tRNA synthetases have been localized in five organisms, and the nucleotide and the amino acid sequences have been established. The lysyl-tRNA synthetase molecules are of average chain lengths among the aminoacyl-tRNA synthetases, which range from about 300 to 1100 amino acids. Lysyl-tRNA synthetases act as dimers; in eukaryotes they can be localized in multienzyme complexes and can contain carbohydrates or lipids. Lysine tRNA is recognized by lysyl-tRNA synthetase via standard identity elements, namely anticodon region and acceptor stem. The aminoacylation follows the standard two-step mechanism. However the accuracy of selecting lysine against the other amino acids is less than average. The first threedimensional structure of a lysyl-tRNA synthetase worked out very recently, using the enzyme from the Escherichia coli lysU gene which binds one molecule of lysine, is similar to those of other class II synthetases. However, none of the reaction steps catalyzed by the enzyme is clarified to atomic resolution. Thus surprising findings might be possible. Lysyl-tRNA synthetase and its precursors as well as its substrates and products are targets and starting points of many regulation circuits, e.g. in multienzyme complex formation and function, dinucleoside polyphosphate synthesis, heat shock regulation, activation or deactivation by phosphorylation/dephosphorylation, inhibition by amino acid analogs, and generation of antibodies against lysyl-tRNA synthetase. None of these pathways is clarified completely.

Amino Acid Sequence↗

PubMed1995

Threonyl-tRNA synthetase.

Threonine contributes to the solubility and reactivity of proteins by its hydroxy group as well as to the formation and stability of the hydrophobic core of proteins by its methyl group. One may assume that the use of this bifunctional and simply structured amino acid was established early in evolution. Whereas the catalytic pathway of threonine activation and transfer into protein does not deviate essentially from those catalyzed by other aminoacyl-tRNA synthetases, the enzyme specific for threonine exhibits several interesting individual properties: its biosynthesis is regulated by feedback mechanisms, it can be selectively inhibited (out of twenty aminoacyl-tRNA synthetases) by the antibiotic borrelidin, and it can be a target for autoantibodies, thus being involved in the course of autoimmune diseases. The enzyme has been isolated from more than ten organisms showing a dimeric nature and molecular masses between 110 and 220 kDa. Additionally, in several of these cases, the gene of threonyl-tRNA synthetase has been localized, cloned and sequenced, exhibiting proteins of 400 to 800 amino acids chain length. More interesting facts can be expected from future research ranging from chemistry and molecular biology to medicine, e.g. by elucidation of the three dimensional structures of threonyl-tRNA synthetases and of their antigenic epitopes, possibly followed by therapeutic use of less antigenic mutant proteins.

Animals↗

PubMed1994

Threonyl-tRNA synthetase from yeast. Discrimination of 19 amino acids in aminoacylation of tRNA(Thr)-C-C-A and tRNA(Thr)-C-C-A(2'NH2).

For discrimination between threonine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for threonine, discrimination factors (D) have been determined from Kca and Km values. The lowest values were found for Cys, Met, Val (D = 70-280), indicating that threonine is only 70-280-times more often esterified to tRNA(Thr)-C-C-A than are these non-cognate compounds at the same amino acid concentrations. The highest D values have been observed for Gly, Pro, Gln, Leu, Phe, and Lys (D = 1000-2000), for the other non-cognate amino acids D values are in the medium range 300-1000. Generally, threonyl-tRNA synthetase is less specific than the class I enzymes specific for Ile, Val, Tyr, Arg, but more specific than the only investigated class II enzyme specific for Lys. In aminoacylation of tRNA(Thr)-C-C-A(2'NH2) discrimination factors D1 are in the range 2-170. From D1 values and AMP-formation stoichiometry, pre-transfer proof-reading factors II1, were determined; post-transfer proof-reading factors II2 were determined from D values and AMP-formation stoichiometry in acylation of tRNA(Thr)-C-C-A. II1 values are in the range 1.8-33, II2 values in the range 1.4-22, thus threonyl-tRNA synthetase shows the highest post-transfer proof-reading activity of six investigated synthetases (specific for Ile, Val, Tyr, Arg, Lys). Initial discrimination factors caused by differences in Gibbs free energies of binding between threonine and non-cognate amino acids have been calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors (I1 and I2) have been determined which can be related to hydrophobic interaction forces depending on accessible surface areas of the amino acids. The threonine side chain must be bound by hydrophobic forces and two hydrogen bonds. In contrast to proof-reading factors obtained with the synthetases specific for Ile, Val, Tyr, Arg, and Lys, proof-reading factors II1 and II2 obtained with threonyl-tRNA synthetase are also related to hydrophobic interaction of the amino acid side chains and the enzyme. Threonyl-tRNA synthetase examines side chain structures of amino acids in the four postulated recognition steps, for each step the enzyme uses special distinct structures or conformations of the binding cleft.

Adenosine Triphosphate↗

PubMed1994

Nucleotide binding by multienzyme peptide synthetases.

Peptide synthetases consist of linearly arranged catalytic units, which by sequence alignment show equally spaced amino-acid-activating segments/modules of 600-700 amino acid residues. The consensus sequence comprises a new class of sequence motifs which are shared by some carboxyl-activating enzymes, but which do not occur in aminoacyl-tRNA synthetases. The catalytic properties of peptide synthetases with respect to the nucleotide substrate were investigated by enzyme kinetic studies. In the activation reaction ATP may be substituted by 2'-deoxy-ATP (dATP) and 7-deazaadenosine 5'-triphosphate, substrate analogues which are not recognised by many aminoacyl-tRNA synthetases, and may thus prove useful alternative substrates in the detection of peptide synthetases within complex protein mixtures. ATP derivatives substituted at C2 are substrates, while those substituted at C8 are not, indicating a preference for the anti-conformation in substrate binding. Kinetic studies revealed that coenzyme A is a non-competitive inhibitor of the activation reaction, suggesting the presence of a second nucleotide binding site which accommodates nucleotides with phosphate in the C2' or C3' position. This substrate and inhibition profile is markedly different from that of aminoacyl-tRNA synthetases and indicative of a separate homogeneous family of carboxyl-activating enzymes.

Adenosine Triphosphate↗

PubMed1992

Lysyl-tRNA synthetase from yeast. Discrimination of amino acids by native and phosphorylated species.

Discrimination factors (D) which are characteristic for discrimination between lysine and 19 naturally occurring non-cognate amino acids have been determined from kcat and Km values for native and phosphorylated lysyl-tRNA synthetases from yeast. Generally, both species of this class II aminoacyl-tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90-1700, D values of the phosphorylated species in the range 40-770. The phosphorylated enzyme acts faster and less accurately. In aminoacylation of tRNALys-C-C-A(2'NH2) discrimination factors D1 vary over 30-980 for the native and over 8-300 for the phosphorylated enzyme. From AMP formation stoichiometry and D1 values pretransfer proof-reading factors (II1) of 1.1-56 were calculated for for the native enzyme, factors of 1.0-44 for the phosphorylated species. Post-transfer proof-reading factors (II2) were calculated from D values and AMP formation stoichiometry in acylation of tRNALys-C-C-A. Pretransfer proof-reading is the main correction step, posttransfer proof-reading is less effective or negligible (II2 approximately 1-8). Initial discrimination factors (I), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (delta delta GI), were calculated from discrimination and proof-reading factors. In contrast to class I synthetases, for lysyl-tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three-step process instead of the four-step recognition observed for the class I synthetases. Plots of delta delta GI values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical 'stopper model' of these sites.

Acylation↗

PubMed1990

Valyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Val)-C-C-A and tRNA(Val)-C-C-A(3'NH2).

For discrimination between valine and the 19 naturally occurring noncognate amino acids, as well as between valine and 2-amino-isobutyric acid by valyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values in aminoacylation of tRNA(Val)-C-C-A. The lowest values were found for Trp, Ser, Cys, Lys, Met and Thr (D = 90-870), showing that valine is 90-870 times more frequently attached to tRNA(Val)-C-C-A than the noncognate amino acids at the same amino acid concentrations. The other amino acids exhibit D values between 1,100 and 6200. Generally, valyl-tRNA synthetase is considerably less specific than isoleucyl-tRNA synthetase, but this may be partly compensated in the cell by valine concentrations higher than those of noncognate acids. In aminoacylation of tRNA(Val)-C-C-A(3'NH2) discrimination factors D1 are in the range of 40-1260. From D1 values and AMP formation stoichiometry, pretransfer proof-reading factors pi 1 were determined: post-transfer proof-reading factors II 2 were determined from D values and AMP formation stoichiometry in acylation of tRNA(Val)-C-C-A. II 1 values (7-168) show that pretransfer proof-reading is the main correction step, post-transfer proof-reading (II 2 approximately 1-7) is less effective and in some cases negligible. Initial discrimination factors were calculated from discrimination and proof-reading factors according to a two-step binding process. These factors, due to different Gibbs free energies of binding can be related to hydrophobic interaction forces, and a hypothetical 'stopper' model of the amino-acid-binding site is discussed.

Amino Acid Sequence↗

PubMed1989

Arginyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Arg)-C-C-A and tRNA(Arg)-C-C-A(3'NH2).

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNA(Arg)-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 times more often incorporated into tRNA(Arg)-C-C-A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10,000 and 60,000. In aminoacylation of tRNA(Arg)-C-C-A(3'NH2) discrimination factors D1 are in the range 10-600. From these values and AMP formation stoichiometry, pretransfer proof-reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNA(Arg)-C-C-A, posttransfer proof-reading factors II2 could be calculated, II1 values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (II2 approximately 1-10) plays a marginal role. Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors. According to a two-step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical 'stopper' model of the amino acid recognition site is discussed.

Amino Acyl-tRNA Synthetases↗

PubMed1989

Mechanisms of aminoacyl-tRNA synthetases: a critical consideration of recent results.

During the last 10 years intensive and detailed studies on mechanisms and specificities of aminoacyl-tRNA synthetases have been carried out. Physical measurements, chemical modification of substrates, site-directed mutagenesis, and determination of kinetic parameters in misacylation reactions with noncognate amino acids have provided extensive knowledge which is now considered critically for its consistency. A common picture emerges: (1) The enzymes work with different catalytic cycles, kinetic constants, and specificities under different assay conditions. (2) Chemical modifications of substrates can have comparable influence on catalysis as can changes in assay conditions. (3) All enzymes show a specificity for the 2'- or 3'-position of the tRNA. (4) Hydrolytic proofreading is achieved in a pre- and a posttransfer process. In most cases pretransfer proofreading is the main step; posttransfer proofreading is often marginal. (5) Initial discrimination of substrates takes place in a two-step binding process. For some investigated enzymes, initial discrimination factors were found to depend on hydrophobic interaction and hydrogen bonds. (6) The overall recognition of amino acids is achieved in a process of at least four steps. At present, only a rough overall picture of aminoacyl-tRNA synthetase action can be given.

Amino Acyl-tRNA Synthetases↗

PubMed1989

Isoleucyl-tRNA synthetase from baker's yeast. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-3'dA; role of terminal hydroxyl groups aminoacylation of tRNA(Ile)-C-C-A.

Specificity with regard to amino acids in aminoacylation of tRNA(Ile)-C-C-3'dA by isoleucyl-tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values. The lowest values are observed for Cys, Val, His, and Trp (D2 = 180-1700), indicating that at same amino acid concentrations isoleucine is 180-1700 times more attached to tRNA(Ile)-C-C-3'dA. The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2 = 10,000-30,000). D2 values of the other amino acids are in the range of 2000-10,000. Recognition of most amino acids is achieved in a four-step process. Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof-reading steps occur on the pre- and the post-transfer stage. For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post-transfer proof-reading is negligible. As a special case in discrimination of valine, one initial discrimination step and the post-transfer proof-reading step are lacking. The role of the terminal hydroxyl groups of the tRNA for post-transfer proof-reading is assigned to a simple neighbouring group effect. No preference for the 2' or 3' position in proof-reading can be postulated.

Amino Acyl-tRNA Synthetases↗

PubMed1988

Tyrosyl-tRNA synthetase from baker's yeast. Order of substrate addition, discrimination of 20 amino acids in aminoacylation of tRNATyr-C-C-A and tRNATyr-C-C-A(3'NH2).

The order of substrate addition to tyrosyl-tRNA synthetase from baker's yeast was investigated by bisubstrate kinetics, product inhibition and inhibition by dead-end inhibitors. The kinetic patterns are consistent with a random bi-uni uni-bi ping-pong mechanism. Substrate specificity with regard to ATP analogs shows that the hydroxyl groups of the ribose moiety and the amino group in position 6 of the base are essential for recognition of ATP as substrate. Specificity with regard to amino acids is characterized by discrimination factors D which are calculated from kcat and Km values obtained in aminoacylation of tRNATyr-C-C-A. The lowest values are observed for Cys, Phe, Trp (D = 28,000-40,000), showing that, at the same amino acid concentrations, tyrosine is 28,000-40,000 times more often attached to tRNATyr-C-C-A than the noncognate amino acids. With Gly, Ala and Ser no misacylation could be detected (D greater than 500,000); D values of the other amino acids are in the range of 100,000-500,000. Lower specificity is observed in aminoacylation of the modified substrate tRNATyr-C-C-A(3'NH2) (D1 = 500-55,000). From kinetic constants and AMP-formation stoichiometry observed in aminoacylation of this tRNA species, as well as in acylating tRNATyr-C-C-A hydrolytic proof-reading factors could be calculated for a pretransfer (II 1) and a post-transfer (II 2) proof-reading step. The observed values of II 1 = 12-280 show that pretransfer proof-reading is the main correction step whereas post-transfer proof-reading is marginal for most amino acids (II 2 = 1-2). Initial discrimination factors caused by differences in Gibbs free energies of binding between tyrosine and noncognate amino acids are calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors (I1 and I2) are determined which can be related to hydrophobic interaction forces. The tyrosine side chain is bound by hydrophobic forces and hydrogen bonds formed by its hydroxyl group. A hypothetical model of the amino acid binding site is discussed and compared with results of X-ray analysis of the enzyme from Bacillus stearothermophilus.

Acylation↗

PubMed1988

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.

For discrimination between isoleucine and 19 other amino acids by isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600, discrimination factors D have been determined from kcat and Km values in amino-acylation of cognate tRNA(Ile)-C-C-A. Factors D are also products of initial discrimination factors I' and proof-reading factors II'; D = I' II'. Factors II' were calculated from AMP formation stoichiometries and factors I' as quotients of D and II'; I' = D/II'. II' is considered as a product of a pre- and post-transfer proof-reading factor (II' = II1II2), I' as a product of initial discrimination factors I1 and I2 which are due to two steps of initial discrimination. With the yeast enzyme the highest accuracy is achieved in discrimination between isoleucine and valine (D = 38,000); other D values in a high range (10,000-20,000) are observed for Gly, Ser, Thr, Leu and Met; the lowest factors D belong to Cys, Asp, Asn and Trp (300-700); the remaining amino acids are discriminated with medium D values (1000-10,000). Discrimination factors D observed for isoleucyl-tRNA synthetase from E. coli are on average 2-3 times higher than for the yeast enzyme. Highest values were found in discrimination against Gly, Ala and Val (60,000-72,000), the lowest values for Cys, Arg and Trp (600-3000); the other amino acids exhibit D values between 20,000 and 50,000. Initial discrimination factors can be related to hydrophobic interaction forces between the substrates and the enzyme; a hypothetical model of the amino acid binding site is discussed.

Acylation↗