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

Abstract

The APA1 gene in Saccharomyces cerevisiae encodes Ap4A phosphorylase I, the catabolic enzyme for diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). APA1 has been inserted into a multicopy plasmid and into a centromeric plasmid with a GAL1 promoter. Enhanced expression of APA1 via the plasmids resulted in 10- and 90-fold increases in Ap4A phosphorylase activity, respectively, as assayed in vitro. However, the intracellular concentration of Ap4A exhibited increases of 2- and 15-fold, respectively, from the two different plasmids. Intracellular Ap4A increased 3- to 20-fold during growth on galactose of a transformant with APA1 under the control of the GAL1 promoter. Intracellular adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G) also increased in the transformant under these conditions. The chromosomal locus of APA1 has been disrupted in a haploid strain. The Ap4A phosphorylase activity decreased by 80% and the intracellular Ap4A concentration increased by a factor of five in the null mutant. These results with the null mutant agree with previous results reported by Plateau et al. (P. Plateau, M. Fromant, J.-M. Schmitter, J.-M. Buhler, and S. Blancquet, J. Bacteriol. 171:6437-6445, 1989). The paradoxical increase in Ap4A upon enhanced expression of APA1 indicates that the metabolic consequences of altered gene expression may be more complex than indicated solely by assay of enzymatic activity of the gene product.

Disruption and overexpression of the Schizosaccharomyces pombe aph1 gene and the effects on intracellular diadenosine 5′,5'''-P1,P4-tetraphosphate (Ap4A), ATP and ADP concentrations

“…Upon disruption of APA1, which encodes Ap % A phosphorylase I [9], the concentration of intracellular Ap % A in Sacch. cere isiae increases by a factor of 3-5 [10,20], while disruption of APA2 causes no detectable change in the intracellular Ap % A concentration [10]. However, disruption of both phosphorylase genes leads to a 38-fold increase in intracellular Ap % A concentration [10].…”

“…Previously reported values in Sacch. cere isiae are 3.6 pmol of Ap % A\mg of protein [11,21] and 0.58 pmol of Ap % A\mg of protein [20]. These values can be estimated as concentrations of 310 nM and 50 nM Ap % A respectively, using cell parameters for Sacch.…”

“…When the cells reached the appropriate density, three independent samples (50 or 100 ml each) from each culture were removed and harvested by rapid filtration on 1.2 µm-pore-size Millipore filters, suspended in 5 % (w\v) trichloroacetic acid containing 2 pmol of [$H]Ap % A as an internal standard, and processed for measurement of Ap % A as described previously [20]. One 1-ml rinse of the filter with 5 % (w\v) trichloroacetic acid was added to the resuspended sample before sonication, and the sonicated trichloroacetic acid extracts sat on ice for at least 45 min before centrifugation (43 000 g, 30 min) to obtain trichloroacetic acid supernatants and crude pellets.…”

“…Previous work attempting to achieve a similar goal in Sacch. cere isiae resulted in a paradoxical increase in intracellular Ap % A concentration upon overexpression of Ap % A phosphorylase I, in spite of a maximal 90-fold increase in phosphorylase activity in itro [11,20]. Mechulam et al [44] demonstrated that overexpression of E. coli apaH, which encodes a symmetrical Ap % A hydrolase, decreased the intracellular Ap % A concentration in E. coli by a factor of 10.…”

Control of dinucleoside polyphosphates by the FHIT-homologous HNT2 gene, adenine biosynthesis and heat shock in Saccharomyces cerevisiae

“…In contrast, hnt2ΔADE2 strains achieved a 3.7-fold higher level of AppppN than HNT2 ADE2 strains. Larger increases in AppppN concentrations have been seen with disruption of Apa1 and Apa2, the diadenosine tetraphosphate phosphorylases in S. cerevisiae [34,35], indicating that they have a more significant role in controlling AppppN levels than does Hnt2. In the case of disruption of the Fhit and Hnt2-homologous aph1 gene in S. pombe , which encodes an enzyme relatively specific for a AppppA [20], a 290-fold increase in AppppA concentration was observed [22].…”