Adenylation substrates

An RNA Diels-Alderase ribozyme recently developed that catalyses the formation of carbon-carbon bonds between a tethered anthracene diene and a biotinylated maleimide dienophile. The ribozyme active site has been further characterised by chemical substitution of the diene and dienophile. It was shown that the diene must be an anthracene, and substitution only at specific sites is permitted. The dienophile must be a maleimide with an unsubstituted double bond. The RNA-diene interaction was found to be governed preferentially by stacking interactions. A ribozyme has been selected that catalyses the synthesis of dipeptides using an aminoacyl-adenylate substrate. The ribozyme catalysed the formation of 30 different dipeptides, many with rates similar to that of the Met-Phe dipeptide used in the selection process. 

An artificial ribozyme mimics this translation step of the ribosome. The spedlity of this selected ribozyme is based on the recognition of an adenosine moiety of the amino acid ester and allows the utilization of leucine- and phenylalanine- as weU as methionine-derivatized substrates. This tolerance for various amino acids indicates the possibility of selecting more general ribozymes for protein synthesis. Furthermore, a related ribozyme efficiently catalyses the synthesis of -30 different dipeptides from an aminoacyl-adenylate substrate. Ribozyme-mediated synthesis of uncoded peptides might have been an important step in the transition from a RNA to a peptide world before the anergence of the ribosome. ... 

For luciferin, a firefly luciferase cosubstrate, another method of retention has been evaluated which consisted of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a polymeric matrix31. Using the suitable co-immobilized enzymes (adenylate kinase and creatine kinase), the three adenylic nucleotides (ATP, ADP and AMP) could be assayed continuously and reproducibly with a selfcontainment working time of 3 h. 

Mg2+ is competitive with the Li+ inhibition of both postreceptor G-protein stimulation [140], and direct stimulation of adenylate cyclase [141]. Li+ inhibits Mn2+-stimulated adenylate cyclase activity in membranes in the presence, but not in the absence, of calmodulin. Since, Mn2+ can replace Ca2+ in activating calmodulin, it is likely that the observed inhibition is that of the Mn2+-dependent calmodulin stimulation of the enzyme. In the absence of calmodulin, stimulation of adenylate cyclase is probably due to substitution of Mn2+ for Mg2+ in the substrate, MnATP2+ and this is unaffected by Li+. 

With isotopes it has been possible to show that all enzyme-catalyzed reactions are stereospecific. Before the availability of isotopes, there was no way of testing this generalization. Of course there are some apparent exceptions to prove the rule. Bently has listed a considerable number (2>, Table XIII, Chapter 6). The most interesting one to me seems to be luciferase, but that is an exception that isn t an exception. Thus, the enzyme luciferase acts on its substrate luciferin (2), in the presence of ATP and O2, to oxidize the luciferin to oxyluciferin (3). The reaction consists of an initial activation of the substrate by ATP to give luciferyl adenylate, after which the oxidation takes place. When the natural enantiomer (synthesized from D-cysteine) is activated and oxidized, light is emitted. The other enantiomer is also acted on by the enzyme, and is converted to the adenylate, but oxyluciferin is not formed, and there is no bioluminescence 37,38,38a)... 

The structure of the complex between native enzyme and tyrosyl adenylate has been elucidated (Rubin and Blow, 1981). Tyrosyl adenylate is one of the products of the first step in the enzyme mechanism (47), and is the substrate for the second step of the reaction (48). Eleven possible hydrogen bonds are identified and the structure is given in [88]. Previously, the structure of the complex between the enzyme and the competitive inhibitor tyrosinyl adenylate had been clarified (Monteilhet and Blow, 1978). 

One of the important consequences of studying catalysis by mutant enzymes in comparison with wild-type enzymes is the possibility of identifying residues involved in catalysis that are not apparent from crystal structure determinations. This has been usefully applied (Fersht et al., 1988) to the tyrosine activation step in tyrosine tRNA synthetase (47) and (49). The residues Lys-82, Arg-86, Lys-230 and Lys-233 were replaced by alanine. Each mutation was studied in turn, and comparison with the wild-type enzyme revealed that each mutant was substantially less effective in catalysing formation of tyrosyl adenylate. Kinetic studies showed that these residues interact with the transition state for formation of tyrosyl adenylate and pyrophosphate from tyrosine and ATP and have relatively minor effects on the binding of tyrosine and tyrosyl adenylate. However, the crystal structures of the tyrosine-enzyme complex (Brick and Blow, 1987) and tyrosyl adenylate complex (Rubin and Blow, 1981) show that the residues Lys-82 and Arg-86 are on one side of the substrate-binding site and Lys-230 and Lys-233 are on the opposite side. It would be concluded from the crystal structures that not all four residues could be simultaneously involved in the catalytic process. Movement of one pair of residues close to the substrate moves the other pair of residues away. It is therefore concluded from the kinetic effects observed for the mutants that, in the wild-type enzyme, formation of the transition state for the reaction involves a conformational change to a structure which differs from the enzyme structure in the complex with tyrosine or tyrosine adenylate. The induced fit to the transition-state structure must allow interaction with all four residues simultaneously. 

Asn-tRNA formation in P. aeruginosa involves an ND-AspRS, which forms both Asp-tRNA (direct pathway for Asp) and Asp-tRNA (indirect pathway for Asn). L-Aspartol adenylate (15) inhibits aspartylation of tRNA f" (i) = 41 pmolP ) more efficiently than that of tRNA (A) = 215 pmolffi ), the other natural tRNA substrate of this enzyme. " ... 

Recently, bacterial NRPS modules with the organization of A-KR-PCP have been discovered in the valino-mycin and cereulide synthetases. The A domains of these modules selectively activate a-keto acids. After the resulting adenylate is transferred to the PCP domain, the a-ketoacyl- -PCP intermediate is reduced to a PCP-bound, a-hydroxythioester by the KR domain. These domains use NAD(P)H as a cofactor and are inserted into A domains between two conserved core motifs analogous to MT domains. Their substrate specificity differs from that of polyketide synthase KR domains, which reduce /3-ketoacyl substrates. Similar fungal NRPSs, such as beauvericin synthetase, utilize A domains that selectively activate a-hydroxy acids. These molecules are thought to be obtained using an in trans KR domain, which directly reduces the necessary, soluble a-keto acid. 

Figure 16 (a) Structures of adenylation domain intermediates and inhibitors aminoacyl-sulfamoyl adenosine (AMS) and cisoid -like macrocyclic inhibitor, (b) Alkyne-functionalized chemical probe for NRPS A and PCP domains, (c) Structure of aminoacyl PCP, SNAC substrate analogue, and hydrolytically stable phosphopantetheinyl analogue, (d) Structure of vinylsulfonamide probe. R represents a peptide component and R an amino acid side chain. 

The scheme (Fig. 15.1) thus explained the production of both sulfate and sulfur in equimolar amounts from thiosulfate oxidation. In showing adenylylsulfate as an intermediate, it also provided a feasible route for the conservation of energy from sulfite oxidation by a substrate-level phosphorylation mechanism, in which ADP sulfurylase and adenylate kinase give rise to ATP ... 

Although adenylic acid deaminase is a well-known enzyme that has been isolated in crystalline form, little work has been reported on its substrate specificity evidence for the deamination of 4-aminopyrazolo[3,4-d]pyrimidine ribonucleotide has been presented [118], but it is not known if it catabolizes any of the other intracellularly formed adenylic acid analogues. 

The conversion of inosinic acid to adenylic acid is a two-step process [65] The first step, the conversion of inosinic acid to adenylosuccinic acid, is mediated by adenylosuccinate synthetase [65], which is inhibited by 6-mercaptopurine ribonucleotide [309-311] in a non-competitive manner, although the exact nature of this inhibition is not known [67]. The second step of the sequence, the conversion of adenylosuccinic acid to adenylic acid, is also inhibited by 6-mercaptopurine ribonucleotide [66, 311], and in this case the inhibition is competitive with respect to the substrate [67]. 

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