O. Goerlich, R. Foeckler, E. Holler
Aug 1, 1982
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2
Influential Citations
140
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Journal
European journal of biochemistry
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.