Fructose 1,6-diphosphatase from Rhodopseudomonas palustris. I. Purification and properties

The fructose 1,6-diphosphatase (d-fructose 1,6-diphosphate I-phosphohydrolase, EC 3.1.3.11) of Rhodopseudomonas palustris has been purified 800-fold and found to have a molecular weight of approximately 130,000. Enzyme stored in the cold in the presence of Mn²⁺ ions for longer than 3 weeks dissociates into fully catalytically active dimers of molecular weight of about 65,000. The enzyme was shown to have optimal activity at pH 8.5 and demonstrated an absolute requirement for divalent cations which could be satisfied by Mn²⁺ or Mg²⁺. Maximal catalytic activity in the presence of Mg²⁺ was only 27% of the maximum activity observed with Mn²⁺. Univalent cations such as Li⁺, Na⁺, K⁺, $\text{NH}{}_4{}^{\mathrm{+}}$$\text{NH}{}_4{}^{\mathrm{+}}$, and Cs⁺ were found to inhibit the enzyme to varying degrees. Enzymatic activity was activated fourfold by sulfhydryl reagents such as reduced glutathione and dithiothreitol. p-Hydroxymercuribenzoate completely inactivated the enzyme. Only the cofactor Mn²⁺ offered protection against inhibition by this reagent. The enzyme was inactivated 65% after heating for 1 min at 50 °C, whereas in the presence of 0.3 mm Mn²⁺ only 12% of the catalytic activity was lost after 1 min. The K_m values for fructose 1,6-diphosphate and Mn²⁺ were calculated to be 4.5 × 10⁻⁵m and 2.2 × 10⁻⁵m, respectively. Sedoheptulose 1,7-diphosphate was cleaved at 22.4% of the rate of hydrolysis of fructose 1,6-diphosphate and has a K_m of 30 × 10⁻⁵m. Fructose 1,6-diphosphatase activity was competitively inhibited by sedoheptulose 1,7-diphosphate with the K_I calculated to be 29 × 10⁻⁵m. The enzyme was inactive toward all other substrates tested.