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 homodimeric 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 sixstranded antiparallel ␤-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 ␣/␤ 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.

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 k,,, and K , values obtained in aminoacylation of tRNATY'-C-C-A. The lowest values are observed for Cys, Phe, Trp (D = 28000-40000), showing that, at the same amino acid concentrations, tyrosine is 28000-40000 times more often attached to tRNATY'-C-C-A than the noncognate amino acids. With Gly, Ala and Ser no misacylation could be detected (D > 500000); D values of the other amino acids are in the range of 100000-500000.Lower specificity is observed in aminoacylation of the modified substrate tRNATYr-C-C-A(3'NH2) ( D , = 500 -55000). 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 ( H I ) and a post-transfer (n,) proof-reading step. The observed values of n , = 12-280 show that pretransfer proof-reading is the main correction step whereas post-transfer proof-reading is marginal for most amino acids (n, = 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 ( I , and I , ) 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 .strurothcrmophilus.Tyrosyl-tRNA synthetases represent a group of aminoacyl-tRNA synthetases which has been investigated very intensively and detailed. Complete primary structures are known for the enzymes from Escherichia Cali and Bacillus stenrothermophilus [l, 21, exhibiting an amino acid sequence homology of 56% [2]. Tyrosyl-tRNA synthetase from B. strarothermophilus was crystallized and analyzed by X-ray diffraction as free enzyme [3] and complexed with tyrosyladenylate [4], which is the enzyme-bound product of the first reaction step in the aminoacylation reaction : E + ATP + Tyr + E . Tyr-AMP + PP, E . Tyr-AMP + tRNA + E + Tyr-tRNA + AMP.From X-ray analyses several hydrogen bonds between amino acid side chains of the enzyme and tyrosyladenylate were postulated [5, 61 and a considerable number of mutant enCorrespondence to W. Freist, Abteihng Chemie,

A set of 24 ATP analogs modified at various positions of the ATP molecule was used for mapping the ATP-binding site in the free catalytic subunit (C) of CAMP-dependent protein kinase (type I). Ki values for these analogs (of which 23 were shown to be competitive with ATP) were measured and compared with Ki values previously obtained for the same set of analogs upon binding to the undissociated form of the enzyme (R2C2). It was found that modifications at the adenine part of ATP bring about a considerable reduction in affinity between C and the resulting analog. The other parts of the ATP molecule play a less important, though definite, role in the binding of this nucleotide to C. By measuring the effect of each given modification in ATP on its binding to C, and comparing the effect of this modification on the binding of the same analog to R2C2, it was possible to obtain 'specificity profiles' for both forms of the kinase. Using such profiles it is shown that the adenine-binding subsite in C may well coincide with the adenine-binding subsite in R2C2. Two plausible models describing the spatial relationship between the ATP sites in C and in R2C2 are proposed.In spite of being catalytically inactive, the undissociated form of CAMP-dependent protein kinase (type I, from rabbit muscle) is known to possess a highaffinity site for ATP (& % 50 nM [l]). It was previously shown [2] that this ATP site does not overlap the cAMP sites on the regulatory subunit of the undissociated enzyme since (a) ATP and cAMP do not compete with each other for binding to the cAMP sites and (b) a variety of substitutions in ATP affect the binding of the resulting analogs to the undissociated enzyme in a way which is different from the way parallel substitutions in cAMP affect the binding of these analogs to the same protein. Furthermore, it was pointed out [2] that the high-affinity ATP site in the undissociated enzyme has some features in common with ATP sites of other phosphotransferase enzymes such as .phosphoglycerokinase and tRNA synthetases [3-71, raising the possibility that this ATP site may overlap the ATP site residing in the catalytic subunit of the enzyme. We wish to report here the K i values for the binding of 24 ATP analogs to the pure catalytic subunit, and compare these values with the Ki values

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 carboxylactivating 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 carboxylactivating enzymes.Amino acids can be incorporated into peptides by two peptide-forming systems, the ribosomal system and the nonribosomal multienzymic system. In the ribosomal system, amino acids are activated by aminoacyl-tRNA synthetases as tRNA esters and peptide bond formation is directed by the ribosome. In the nonribosomal system, amino acids are activated on multienzymes also directing peptide bond formation. We looked for possible common features in the primary structure between the two amino-acid-activating systems. In both cases, amino acids are activated by the energy derived from hydrolysis of an ATP a-P linkage.Linear and cyclic low-molecular-mass peptides, containing non-protein constituents like hydroxy and D-amino acids, are usually produced by peptide synthetases, multienzymes employing the thiotemplate mechanism [l 1. These enzymes range in molecular mass between 123 -1400 kDa [2]. So far, a number of peptide synthetases have been purified and characterised and several amino acid sequences have been published [3 -211. Analysis of the primary structures shows that they are organised in highly conserved and repeated functional units. These structural features support the proposed mechanism whereby amino acids activated as aminoacyladenylates at different specific sites on the multienzyme are subsequently linked into the peptide chain [22]. The Abbreviations. ACV, &(L-a-aminoadipyl)-L-cysteinyl-D-valine;RTP, purineriboside S'-triphosphate; 7-deazaATP, 7-deazaadenosine 5'-triphosphate. Surprisingly, even though peptide synthetases and aminoacyl-tRNA synthetases catalyse similar reactions, there are no significant sequence similarities. Therefore, if the peptide t...

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.