Adenyl formation

In case of equilibrium, addition of labeled PPi yields labeled ATP and this product can be employed to detect NRPS or related enzymes. The respective reaction rates provide information on adenylate formation/pyrophosphoiylysis, apparent Km of substrates and substrate analogs, and with some enzyme kinetic efforts, substrate affinities and the patterns of substrate binding may be deduced. The ease and the sensitivity of the procedure makes it the primary method of investigation of NRPS substrate specificity. 

Following structure-activity studies, the adenylate is thought to be stabilized within a cleft formed between the two subdomains of the activation domain [41,58], The rate is thus related to the formation, presence, and stability of this mixed anhydride with respect to PPi, and at high MgATP2 concentrations, with respect to ATP in the formation of diadenosine tetraphosphate (A2P4). Thus a high rate of the amino acid-dependent isotope exchange does not necessarily reflect the efficiency of adenylate formation, and certainly not the efficiency of incorporation of an amino acid into peptidyl intermediates or the final product. 

A fairly large number of potential amino acids has been assayed for adenylate formation. These amino acids can be grouped into compounds presumably being processed by just one of the three domains, or by several of the domains. Some of the data have been compiled in Table 4, and the main questions to be addressed are (1) Do we find significant differences in fungal and bacterial ACV... 

Among activated forms of amino acids, mixed anhydrides with inorganic phosphate or phosphate esters require a special discussion because they are universally involved in peptide biosynthesis through the ribosomal and non-ribosomal pathways. These mixed anhydrides have stimulated studies in prebiotic chemistry very early in the history of this field. Amino acyl adenylates 18c have been shown to polymerize in solution [159,160] and in the presence of clays [139]. However, their participation as major activated amino acid species to the prebiotic formation of peptides from amino acids is unlikely for at least two reasons. Firstly, amino acid adenylates that have a significant lifetime in aqueous solution become very unstable as soon as either CO2 or bicarbonate is present at millimolar concentration [137]. Lacey and coworkers [161] were therefore conduced to consider that CO2 was absent in the primitive atmosphere for aminoacyl adenylate to have a sufficient lifetime and then to allow for the emergence of the modern process of amino acid activation and of the translation apparatus. But this proposition is unlikely, as shown by the analysis of geological records in favor of CO2 contents in the atmosphere higher than present levels [128]. It is also in contradiction with most studies of the evolution of the atmosphere of telluric planets [30,32], Secondly, there is no prebiotic pathway available for adenylate formation and ATP proved to be inefficient in this reaction [162]. 

T. Imanaka, J. C. Thierry, D. Moras, Crystal structure of aspartyl-tRNA synthetase from Pyrococcus kodakaraensis KOD archaeon specificity and catalytic mechanism of adenylate formation, EMBO J. 1998, 17(17), 5227-5237. 

Figure 11.14 Amino acylation mechanisms catalyzed by aminoacyl-tRNA synthetases The two classes of aminoacyl-tRNA synthetases (aRS s) differ in the site of aminoacylation. Class I aRS s aminoacylate 2 -OH whereas class 11 aRS s add amino acids to 3 -OH of the terminal ribose of the 3 -terminal CCA of cognate tRNA. Magnesinm ions complexed with ATP to enter the active site of aRS may play a dual role in the activation step by both stabilizing the conformation of the ATP (Mg ion bridges the P- and y-phosphates) and participating in adenylate formation (second Mg is found between a- and P-phosphates in some aRS s). In class I aRS, both Lys of MSK and His of HIGH stabilize the bipyramidal oxyphosphorane transition state while R of motif 2 in class II aRS participates in the stabilization of the putative pentacoordinate transition state. The resulting mixed anhydride aminoacyl adenylate is held by the enzyme for the next reaction, i.e. the attack by the 2 -OH (class I) or 3 -OH (class II) of the terminal adenosine at the carbonyl of the aminoacyl adenylate. The amino acid then becomes esterified to the cognate tRNA.

The direct route of acyl coenzyme A synthesis from a free carboxylic acid is catalysed by a group of nucleoside triphosphate-requiring en mes, collectively known as thiokinases. The general mechanism, as exemplified for acetate activation by acetyl thiokinase, proceeds as follows. The carboxylic acid is first activated by acetyl adenylate formation with the displacement of pyrophosphate from ATP. While the initial reaction is fully reversible, subsequent action of pyrophosphatase drives the reaction... 

Figure I Schematic views of reactions involved in peptide biosynthesis. (1) Adenylate formation involving nucleophilic attack of the carboxyl group at the a-phosphate of the MgATP --complex with release of MgPP. - (2) aminoacylation of the pantetheine cofactor by formation of the thio-late anion, attack of the mixed anhydride, and release of AMP (3) tentative view of the peptide bond formation by nucleophilic attack of the aminoacyl-nitrogen at the preceding thioester-car-boxyl, involving deprotonaiion-protonation (4) epimerization of an aminoacyl-thioester, a reaction differing from those catalyzed by the well-characterized amino acid racemases. (Altered from Ref. 13. )...

Figure 3 Peptide synthetase mcxlules. A peptide synthetase module (top) can be dissected into submodules catalyiing adenylate formation (AI and AZ), an acyl carrier module (S), and a con densing module (C) condensing modules with epiraerizing function have two additional motifs (Cg). N-methyhransferase modules (M) are inserted between Al and A2. For terminating reactions, a thioesteiasc module (TE) may be added.

The S. chrysormllus enzyme displays dififerent activities in the presence of a number of different benzene carboxylic acids tested as substrates (Table 2). The value of the equilibrium constant of the adenylate formation reaction, was increased when substituents such as an amino-, hydroxyl-, or methyl-groups in the 4-position were present, compared with benzoic acid alone. The same substituents in the 3-position resulted in a less pronounced response of the enzyme, whereas substituents in the Z-position were... 

Reactions (81) and (82) are responsible for the S3mthesis of acetyl C oA (315) (R = —CH ) and reaction (81) is the means by which an amino acid is activated (313, 314). Hydroxamate formation by reaction (83) is nonenzymic and is commonly used to assay acid-adenylate formation (314). 

As discussed earlier, AARSs have core catalytic domains that perform the functions of aminoacyl adenylate formation and transfer of the amino acid to the cognate tRNA. The sequences and structures of these domains also differentiate the enzymes as belonging to Class I or II. In addition to this class-defining active site domain, most AARSs also have one or more appended domains that are unique. These idiosyncratic domains often make specific contacts with recognition elements outside the tRNA acceptor stem, for example, at the anticodon or variable loop of the tRNA molecule (Fig. 4). In addition to the two-domain (or more) organization of the AARS enzymes, tRNAs can also be viewed as modular structures. As mentioned earlier, the acceptor stem and T4 C arm coaxially stack to form one portion of the L-shaped tRNA structure, while the D and anticodon arms stack to make the other tRNA arm (Fig. 2). The acceptor arm makes contacts with the catalytic core of the enzyme and contains the amino acid attachment site, while the anticodon, located on the second arm of the tRNA, is recognized by an appended domain. 

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