Yeast diadenosine 5',5'''-P1,P4-tetraphosphate .alpha.,.beta.-phosphorylase behaves as a dinucleoside tetraphosphate synthetase

Abstract

The diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.

Catabolism of bis(5'-nucleosidyl) tetraphosphates in Saccharomyces cerevisiae

“…Therefore, the mechanisms of Ap4A degradation by the two Ap4A phosphorylases appeared identical. It was also verified that Ap4A could be obtained from ATP and ADP through the reverse reaction of phosphorolysis (8,14). The two Ap4A phosphorylases required the addition of divalent ions to measurably catalyze Ap4A phosphorolysis.…”

“…1 filter paper disks, and the radioactivity was measured in a Beckman LS1801 counter with the scintillation cocktail Picofluor (Packard). In vitro synthesis of Ap4A from 4 mM ATP and 4 mM ADP in the presence of catalytic amounts of Ap4A phosphorylase I or II was assayed as described previously (8).…”

Isolation, characterization, and inactivation of the APA1 gene encoding yeast diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase

“…Initial rates of Ap4A degradation were assayed by a procedure resembling the radioisotopic assay previously established for E. coli Ap4A hydrolase activity measurements (46 30 min, 37°C) to remove residual ATP. Then, Ap4N was quantified in the luciferin-luciferase phosphodiesterase assay described previously (10,40). The cellular concentration of nucleotides was calculated with the assumption that an OD650 of 1 corresponded to 0.4 ,ul of intracellular volume (14).…”

A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae

“…Thus, when the Ap4A phosphorylase activity in strain CGY339 was increased by about 10-fold and 100-fold as measured in vitro, the intracellular Ap4A increased by about 2-fold and 15-fold, respectively, as indicated by the data in Tables 2 and 3 (13,25,38) and thus cannot synthesize Gp4G. Ap4A phosphorylase I can synthesize Ap4A and other dinucleoside tetraphosphates, including Gp4G, at a low pH in vitro (8). However, on the bases of the intracellular pH in growing yeast cells (about 7.2) (10), the intracellular concentrations of ATP, ADP, and Pi as stated by Brevet et al (8), and the measured equilibrium constant of the reaction (8), the equilibrium concentration of Ap4A under intracellular conditions would be about 0.09 ,uM.…”

“…Ap4A phosphorylase I can synthesize Ap4A and other dinucleoside tetraphosphates, including Gp4G, at a low pH in vitro (8). However, on the bases of the intracellular pH in growing yeast cells (about 7.2) (10), the intracellular concentrations of ATP, ADP, and Pi as stated by Brevet et al (8), and the measured equilibrium constant of the reaction (8), the equilibrium concentration of Ap4A under intracellular conditions would be about 0.09 ,uM. Both the basal intracellular concentrations of Ap4A (0.3 to 0.55 ,uM [9,14]) and the elevated levels reported here are significantly higher than this.…”