Mechanism of Synthesis of Adenosine(5′)tetraphospho(5′)adenosine (AppppA) by Aminoacyl‐tRNA Synthetases

Abstract

Synthesis of radioactive adenosine(5')tetraphospho(Sf)adenosine in the presence of aminoacyl-tRNA synthetases of procaryotes or eucaryotes was measured by thin-layer chromatography on poly(ethy1eneimine)-cellulose sheets. Catalysis by the majority of aminoacyl-tRNA synthetases was observed. They were classified into a fast group (specificity for Lys, Phe, His), a slow group (Ser, Ile, Leu, Asp, Tyr, Val) and a non-catalytic group (Trp, Arg). The presence of the cognate amino acid is required.The mechanism was verified by direct synthesis of the tetraphosphate from preformed aminoacyladenylate and by its reverse reaction from chemically synthesized adenosine(5')tetraphospho(5')adenosine. Essentials are as follows. (1) Aminoacyladenylate is synthesized from a first molecule of MgATP. (2) A second ATP adds as substrate, Michaelis-Menten constants being 11 mM and 50 mM in the case of Escherichia coji lysine-specific and phenylalanine-specific enzymes, respectively; these values are two orders of magnitude higher than for the first ATP. (3) This is followed by the synthesis of adenosine(5')tetraphospho(5')adenosine from adenylate and the second ATP; the reaction is rate-determining in catalysis, rate constants being 0.5 s-' and 0.26 s-l respectively. (4) The equilibrium constant, K s 0.02, does not favour the synthesis of the tetraphosphate from ATP; removal of accumulating pyrophosphate by the action of added inorganic pyrophosphatase increases yields substantially. ( 5 ) The presence of Mg2+ ions is essential; in a second role, they can strongly activate the catalytic reaction. (6) Znz+ is a more potent activator, although it is not essential; its effect was to decrease the value of the Michaelis-Menten constant and to increase that of the catalytic rate constant; activation was observed only for a minority of aminoacyl-tRNA synthetases (Phe, Lys).The synthesis of adenosine(5')tetraphospho(5')adenosine and of adenosine(5')triphospho(5')adenosine and its reverse are discussed with regard to possible biological functions.

P1, P3‐bis(5′‐adenosyl)triphosphate (Ap3A) as a substrate and a product of mammalian tryptophanyl‐tRNA synthetase

“…For Ap4A-synthesyzing aaRS, Ap3 A synthesis is a secondary process: aminoacyl adenylate.enzyme complexes react with ADP generated in prokaryotes from Ap4A degradation to 2 ADP (see [2,6] and references therein). Therefore, as TrpRS does not promote Ap4 A synthesis it is unable to synthesize Ap3A through Ap4 A decomposition.…”

“…At the same time, these enzymes are able to utilize at least in vitro [4] Ap4A for the formation of aminoacyl adenylate. Besides Ap4A synthesis, Ap3A formation was reported for the same enzymes from the adenylate.enzyme complexes where ADP was generated in the process of Ap4A hydrolysis [6,7]. No aaRS exhibited Ap3A synthesis independent from Ap4 A formation.…”

“…The lack of ApaA synthesis by TrpRS [6] might be in principle connected with the non-optimal experimental conditions chosen for its detection. Therefore, we decided to examine Ap4A-synthetase activity of TrpRS at variable concentrations of TrpRS ( 1-10/JM), ATP ( 1 -10 mM) and tryptophan (10-2-1 mM).…”

“…It was suggested that these aaRS are also able to catalyze in vivo Ap4A and Ap3A synthesis. ApnA synthesis catalyzed by these aaRS proceeds through the stage of the aminoacyl adenylate, enzyme complex formation [6,7] as does the synthesis of aminoacyl-tRNA. At the same time, these enzymes are able to utilize at least in vitro [4] Ap4A for the formation of aminoacyl adenylate.…”

Do Aminoacyl-tRNA synthetases have biological functions other than in protein biosynthesis?

“…ARSs also catalyze a secondary reaction in which the aminoacyl-AMP reaction intermediate reacts with ATP to produce diadenosine tetraphosphate (Ap 4 A) (27,28). Protein kinase C-dependent phosphorylation of ARSs increases Ap 4 A synthesis by several fold (29) without affecting the aminoacylation reaction.…”