Partial purification and properties of the fructose-1,6-diphosphatase from Acinetobacter lwoffi

Abstract

Fructose-1,6-diphosphatase (FDPase) from Acinetobacter lwoffi ( Mima polymorpha) was partially purified and some of its properties investigated. The enzyme is constitutive for the organism and its levels are not affected by the different growth substrates used. Time study experiments showed no appreciable fluctuations during the different growth phases in any medium. Like most other FDPases studied, the enzyme in Acinetobacter requires a divalent cation like Mg 2+ or Mn 2+, has a high affinity for the substrate, is stimulated by low concentrations of EDTA, has a pH optimum in the alkaline range, and is inhibited by high substrate concentrations. The presence of EDTA in the reaction mixture causes a small shift in the pH optimum in different buffers. The enzyme is strongly inhibited by ATP, ITP, and GTP and to lesser extents by ADP, IDP, and citrate but is relatively insensitive to AMP. However, a lowering of pH results in some enhancement of AMP inhibition. Evidence is presented to show that the treatment of the enzyme with ATP and citrate results in an alteration of the protein leading to reduced affinity for the substrate and consequently reduced activity. At moderate concentrations, the inhibition caused by ATP and citrate together is additive. Three different assay procedures failed to demonstrate any phosphofructokinase activity in crude and partially purified preparations of Acinetobacter lwoffi. It is postulated that modulation of the enzyme by the substrate itself is a major regulatory mechanism in this highly oxidative organism.