PP(i)-dependent phosphofructotransferase (phosphofructokinase) activity in the mollicutes (mycoplasma) Acholeplasma laidlawii

Abstract

A PP(i)-dependent phosphofructotransferase (PP(i)-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1,6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430-fold). PP(i) was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or P(i) could not substitute for PP(i). The PP(i)-dependent reaction (2.0 mM PP(i)) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (≤300 μM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1,6-P2 to F-6-P, required P(i); arsenate could substitute for P(i), but not PP(i) or any other nucleotide tested. The computer-derived kinetic constants (± standard deviation) for the reaction in the PP(i)-driven direction of F-1,6-P2 were as follows: v, 38.9 ± 0.48 mM min-1; K(a(PP(i)), 0.11 ± 0.04 mM; K(b(F-6-P)), 0.65 ± 0.15 mM; and K(ia(PP(i)), 0.39 ± 0.11 mM. A. laidlawii B-PG9 required PP(i) not only for the PP(i)-phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity, a dependency unknown in any other organism. In A. laidlawii B-PG9, the PP(i) requirement may be met by reactions in this organism already known to synthesize PP(i) (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PP(i) instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process.