Pyrophosphat-abhängige ᴅ-Fructose-6-phosphat Phosphotransferase in Rhodospirillaceae / Pyrophosphate-Dependent ᴅ-Fructose-6-phosphate-Phosphotransferase in Rhodospirillaceae

Abstract

Abstract A pyrophosphate-dependent fructose-6 -phosphate phosphotransferase from the photosynthetic bacterium Rhodospirillum rubrum was partially purified and characterized in respect to kinetic and regulatory properties. The enzyme had a molecular weight of about 95 000 dalton and required Mg2+ -ions for catalysis and maintenance of activity. The phosphotransferase was specific for fructose-6 -phosphate (F-6 -P) and inorganic pyrophosphate (PP) as substrates of the fructose-1,6 -bisphosphate-forming reaction (forward reaction). In the phosphate (PO-dependent back reaction, the preferred substrate was fructose-1,6-bisphosphate (FBP). At optimal pH (7.2 for the forward, and 8.6 for the back reaction) the back reaction had a slightly higher Ʋmax than the forward reaction. The substrate-saturation curves of the enzyme were all hyperbolic with intersecting kinetic pattern. The Km-values (in mᴍ) at saturating MgCl2-concentration were: 0.38 (F-6-P); 0.025 (PP); 0.02 (FBP) and 0.82 (Pi). The forward reaction was inhibited by ADP and AMP. The inhibition by ADP (Ki = 0.18 mᴍ) was of the mixed type in respect to F-6-P, but independent of the PP-concentration. The inhibition by AMP (Ki = 0.017 mᴍ) was of a more complex type, because AMP not only decreased the Ʋmax of the F-6-P-or PP-saturation curve, but also increased the Hill-coefficient from nH = 1 to nH = 2.5 of the F-6-P-saturation curve. The inhibition of the back reaction by the two adenylates was less pronounced. ATP (at 2.5 mᴍ), like citrate, inhibited the back reaction only at low MgCl2 concentration (1 mᴍ) indicating that the inhibitory effect was due to the chela­tion of Mg2+. Out of 5 other species of the Rhodospirillaceae tested, the PP-dependent phospho-fructokinase was only shown to be present in Rhodopseudomonas gelatinosa.