Fructose 1, 6-Bisphosphatase from Rabbit Liver. [18O]Phosphate-H2O Exchange as a Probe of the Catalytic Mechanism

Fructose 1, 6-bisphosphatase (FBPase, EC 3.1.3.11, d-fructose-1, 6-bisphosphate 1-phosphohydrolase) has been found to catalyze the solvent exchange of 18O from initially highly enriched inorganic phosphate (P1). The exchange occurs with either Mg2+ or Mn2+ as the cofactor. The exchange proceeds negligibly in the presence of P1 alone but is greatly stimulated by the presence of fructose 6-phosphate, the second product of the FBPase reaction. A theoretical treatment of 18O exchange from P1 is presented, based upon the ability to determine the 18O isotopic distribution in a P1 sample. The analytical technique permits the determination of the percentage of Pi molecules in a sample which contain none, one, two, three, or four 18O atoms per Pi molecule, in addition to the total atom percent 18O of the sample. Comparative data are presented, illustrating the sensitivities and precision of an 18O-shifted 31P NMR technique and the mass spectrometric method, in which the latter is shown to be approximately 102-103 times more sensitive and considerably more precise in the determination of low abundance species. The theoretical treatment defines the conditions under which more than one 18O atom can be exchanged per interaction of a Pi molecule with an enzyme. The quantitative features of exchange time courses, expressed in terms of a rate of exchange/rate of dissociation partition coefficient (kx/koff), are described. Dissociation of Pi from either Mn2+- or Mg2+-FBPase under equilibrium isotope exchange conditions is sufficiently slow so that exchange of more than one 18O can occur per protein-ligand interaction: kx/koff = 1.4-2.0. However, during steady-state hydrolysis of fructose 1, 6-bisphosphate in [18O]H2O, only one oxygen of the Pi produced becomes equilibrated with the solvent, predicting a kx/koff ≤0.01. Such results suggest that either (1) the intermediates involved in equilibrium exchange are different than those involved in hydrolysis or (2) the state of occupancy of the active sites in the enzyme tetramer may control the apparent dissociation rates. However, the observed equilibrium exchange suggests a possible net reversal by FBPase of the hydrolysis reaction.