Zinc(II)-dependent synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate by Escherichia coli and yeast phenylalanyl-tRNA synthetases

Abstract

A new activity of Escherichia coli and yeast phenylalanyl-tRNA synthetases, the conversion adenosine 5' -triphosphate into diadenosine 5' ,5"' -P(1) ,P(4) -tetraphosphate, is reported. This activity is followed by (31)P NMR and chromatography on poly(ethylenimine)-cellulose. It is revealed by the addition of ZnCl2 to a reaction mixture containing the enzyme, ATP-Mg(2+), L-phenylalanine, and pyrophosphatase It reflects the reaction enzyme-bound phenylalanyl adenylate with ATP instead of PPi and strongly depends on the hydrolysis of pyrophosphate in the assay medium. The zinc dependence of this reaction parallels that of the inhibition of tRNA(phe) aminoacylation which is described in the accompanying paper [Mayaux, J. F., & Blanquet, S. (1981) Biochemistry (preceding paper in this issue)]. In the presence of an unlimiting pyrophosphatase activity, diadenosine tetraphosphate synthesis by E. coli and yeast phenylalanyl-tRNA synthetases occurs at maximal rates of 0.5 and 2 s-1, respectively (37 degrees C, pH 7.8, 150 mM KC1, 5 mM ATP, 10 mM MgCl2, 2 mM L-phenylalanine, and 80 muM ZnCl2). Under identical experimental conditions, E coli isoleucyl-, methionyl-, and tyrosyl-tRNA synthetases produce small amounts of diadenosine tetraphosphate at rates 2 or 3 orders of magnitude lower than that achieved by phenylalanyl-tRNA synthetase. In the case of E. coli phenylalanyl-tRNA synthetase, it is shown that the diadenosine tetraphosphate synthetase activity is accompanied by a diadenosinetetraphosphatase activity. This activity, actually supported by phenylalanyl-tRNA synthetase, is responsible for the appearance of ADP in the assay medium. It requires also the presence of both ZnCl2 and L-phenylalanine. The formation of ADP from diadenosine tetraphosphate and its reaction with enzyme-bound aminoacyl adenylate account for the appearance in the reaction mixture of diadenosine 5' ,5"' -P(1) ,P(3)-triphosphate, after that of diadenosine tetraphosphate. The significance of these findings in the context of the role of diadenosine tetraphosphate in controlling cellular growth is discussed.

In vivo levels of diadenosine tetraphosphate and adenosine tetraphospho-guanosine in Physarum polycephalum during the cell cycle and oxidative stress.

“…Ap4A and Ap4G transiently increased three-to sevenfold after 1 h and then decreased concomitantly with an 80% decrease in the level of ATP after 2 h in the presence of dinitrophenol. These results do not support the hypothesis that Ap4A is a positive pleiotypic activator that modulates DNA replication, but they are consistent with the hypothesis proposed for procaryotes that ApA and Ap4G are signal nucleotides or alarmones of oxidative stress (B. R. Bochner Diadenosine 5',5" '-P',P4-tetraphosphate (Ap4A) and other adenosine tetraphosphonucleoside (Ap4N) and adenosine triphosphonucleoside (Ap3N) compounds are synthesized as side reaction products by tRNA synthetases from several organisms, including Physarum polycephalum (6,19,36,40,55). Zamecnik and Stephenson (54) have suggested that Ap4A might serve as a regulatory nucleotide that couples protein synthesis and transcription or replication.…”

Isothermal Titration Calorimetry Reveals a Zinc Ion as an Atomic Switch in the Diadenosine Polyphosphates

“…In particular, we have been interested in the role of Zn 2ϩ in strongly augmenting the aminoacyl-tRNA synthetase synthesis of adenyl dinucleoside polyphosphates (17)(18)(19) and the role of the ion in the heat shock response. Most of the class II aminoacyl-tRNA synthetases are stimulated by Zn 2ϩ in Ap n A synthesis, yet many do not have clear zinc-binding motifs.…”

Drastic rise of intracellular adenosine(5′)tetraphospho(5′)adenosine correlates with onset of DNA synthesis in eukaryotic cells

“…produced in the back reaction of the amino acid activation step of protein synthesis [6] and its biosynthesis can be dramatically stimulated by low concentrations (80 pM) of Zn2+ [7]. The site of its intracellular activity has been shown to be the DNA replicative machinery.…”