Yeast fructose-2,6-bisphosphate 6-phosphatase is encoded by PHO8, the gene for nonspecific repressible alkaline phosphatase.
Yeast fructose-2,6-bisphosphate 6-phosphatase has been purified 7000-fold by heat treatment, poly(ethylene glycol) precipitation, ion-exchange chromatography with Q-Sepharose Fast Flow and Mono Q followed by affinity chromatography with concanavalin-A-Sepharose and gel filtration with Superose 12. The purified dimeric enzyme contains 1.5 mol zinc and 1.3 mol copper/mol subunit. It reacts with fructose 2,6-bisphosphate [Fru(2,6)P2] as well as with p-nitrophenyl phosphate (NpP) showing a pH optimum at pH 6-6.5 with Fru(2,6)P2 [Plankert, U., Purwin, C. & Holzer, H. (1988) FEBS Lett. 239, 69-72] and above pH 9.0 with NpP. The following observations suggest that activity with both substrates depends on the same protein. (a) During 7000-fold purification, the ratio of activity with NpP to that with Fru(2,6)P2 remained constant. (b) The time course of inactivation of enzyme activity in dilute solution at 30 degrees C is similar for both substrates. (c) At increasing temperatures, inactivation of enzyme activity measured with both substrates proceeds at nearly identical rates. (d) Activity with both substrates is found preferentially in the vacuoles. (e) Mutants defective in the nonspecific alkaline phosphatase coded by the PHO8 gene are also defective in Fru(2,6)P2 6-phosphatase activity. (f) A proteinase A mutant, defective in processing and activation of nonspecific alkaline phosphatase coded by the PHO8 gene, also fails to activate Fru(2,6)P2 6-phosphatase.
Plankert
U
U
Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
Purwin
C
C
Holzer
H
H
D000469:Alkaline Phosphatase / Q000235:genetics*; D004794:Enzyme Repression; D005800:Genes, Fungal; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D015003:Yeasts / Q000201:enzymology*
D000469:Alkaline Phosphatase; D010744:Phosphoric Monoester Hydrolases; C085449:fructose-2,6-bisphosphate 6-phosphatase