Synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (AppppA) from adenosine 5'-phosphosulfate and adenosine 5'-triphosphate catalyzed by yeast AppppA phosphorylase

Abstract

A novel way of enzymatic synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), which does not involve aminoacyl-tRNA synthetases, has been discovered. Yeast AppppA alpha, beta-phosphorylase catalyzes irreversible conversion of adenosine 5'-phosphosulfate (APS) and ATP into AppppA according to the equation APS + ATP----AppppA + sulfate. In this reaction, the enzyme exhibits a broad pH optimum (between 6 and 8) and requires Mn2+, Mg2+, or Ca2+ ions for activity, with Mn2+ being twice as effective as Mg2+ or Ca2+ at optimal concentration (0.5 mM). The Km values computed for APS and ATP are 80 microM and 700 microM, respectively. The rate constant for the AppppA synthesis is 3 s-1 (pH 8.0, 30 degrees C, 0.5 mM MgCl2). Some ATP analogues like ppppA, GTP, adenosine 5'-(alpha, beta-methylenetriphosphate), and adenosine 5'-(beta, gamma-methylenetriphosphate), but not dATP, UTP, or CTP, are also substrates for AppppA phosphorylase and accept adenylate from APS with the formation of AppppA, AppppG, Appp(CH2)pA, and App(CH2)ppA, respectively. Functional versatility of yeast AppppA phosphorylase may provide a link between metabolism of AppppA on one hand and metabolism of APS and phosphate on the other and raises the possibility of participation of AppppA in regulation of metabolism of APS and/or inorganic phosphate in yeast.

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

“…150,000 to 200,000 by sucrose gradient centrifugation (18). Such (iii) Finally, this study favors the idea that Ap4A phosphorylase I and the previously described ADP sulfurylase could be a same enzyme, reinforcing the hypothesis of a connection between Ap4A and AMPS metabolism (22). ADP sulfurylase activity has already been detected in various microorganisms, such as Thiobacillus thioparus (43) and Desulfovibrio desulfuricans (44), and in the chloroplasts of higher plants (12).…”

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

“…This result indicates that, in yeast cells, the catabolism of these nucleotides is mainly sustained by Ap4A phosphorylase I and II activities. Consequently, the capacity of Ap4A phosphorylase to synthesize Ap4N or Bp4B', as revealed in vitro (8,16), seems not to apply in vivo, at least under the growth conditions assayed here. This conclusion is reminiscent of the case for E. coli, in which an Ap4N hydrolase ensures the catabolism of the Np4N' nucleotides during exponential growth as well as during stress adaptation (20).…”

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase

“…Reactions (2) and (3) had not been previously described for luciferase in spite of extensive studies carried out on the interchange between the adenylyl moiety of the E-LH2-AMP complex and ATP or PPi [18,191, and the common use of luciferase to measure ATP and Ap4A [20, 211. Here we present the kinetic characteristics of the synthesis of Ap4A and other DNPPs by luciferase and compare these reactions with others reported previously for some aminoacyl-tRNA synthetases [I0 -171 and Ap4A phosphorylase [22]. …”