Fructose 1,6-diphosphatase from Rhodopseudomonas palustris: II. Regulatory properties

Kinetic studies of fructose 1,6-diphosphatase ( d-fructose 1,6-diphosphate I- phosphohydrolase; EC 3.1.3.11) from Rhodopseudomonas palustris has revealed allosteric regulation of the enzyme by fructose 1,6-diphosphate, sedoheptulose 1,7-diphosphate, Mn 2+ ions, GTP, and ATP. Although substrate regulation by fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate are qualitatively similar, the enzyme was observed to interact preferentially with the hexose substrate as judged by the criterion of greater affinity, higher Hill coefficient, and sensitivity to GTP inhibition. Allosteric interactions by fructose 1,6-diphosphate were enhanced by lowering the concentration of Mn 2+ ions or coincubation with the allosteric inhibitor, GTP. Manganese cooperativity was increased by GTP and decreased levels of fructose 1,6-diphosphate. Inhibition studies with freshly prepared enzyme demonstrated that GTP and ATP inhibited enzymatic activity to the same degree. All other nucleoside triphosphates tested were approximately one half as effective. The extent of allosteric interactions exhibited by various modifiers was strictly pH dependent as cooperativity exhibited at neutral hydrogen ion concentration (pH 7.4) was completely abolished under alkaline conditions (pH 8.5). Although shifts in pH did not substantially alter the K m of fructose 1,6-diphosphate and Mn 2+, a 5-fold enhancement of the K I for GTP was observed with a pH increase from 7.4 to 8.5. The data suggest that the physiological significance of the regulation of R. palustris fructose 1,6-diphosphate is that of controlling the Calvin-Bassham carbon-reduction cycle.