BIOCHEMICAL-PROPERTIES OF MUTANT AND WILD-TYPE FRUCTOSE-1,6-BISPHOSPHATASES ARE CONSISTENT WITH THE COUPLING OF INTRASUBUNIT AND INTERSUBUNIT CONFORMATIONAL-CHANGES IN THE T-STATE AND R-STATE TRANSITION

The significance of interactions between AMP domains in recombinant po rcine fructose-1,6-bisphosphatase (FBPase) is explored by site-directe d mutagenesis and kinetic characterization of homogeneous preparations of mutant enzymes. Mutations of Lys(42) ILe(190), and Gly(191) do not perturb the circular dichroism spectra, but have significant effects on ligand binding and mechanisms of cooperativity. The K-m for fructos e 1,6-bisphosphate and the K-i for the competitive inhibitor, fructose a,g-bisphosphate, decreased by as much as 4- and 8-fold, respectively , in the Q32L, K42E, K42T, I190T, and G191A mutants relative to the wi ld-type enzyme, Q32L, unlike the other four mutants, exhibited a 1.7-f old increase in K-cat. Mg2+ binding is sigmoidal for the five mutants as well as for the wild-type enzyme, but the Mg2+ affinities were decr eased (3-22-fold) in mutant FBPases. With the exception of Q32L (8-fol d increase), the 50% inhibiting concentrations of AMP for K42E, K42T, I190T, and G191A were increased over 2,000-fold (>10 mM) relative to t he wild-type enzyme. Most importantly, a loss of AMP cooperativity was found with K42E, K42T, I190T, and G191A. In addition, the mechanism o f AMP inhibition with respect to Mg2+ was changed from competitive to noncompetitive for K42T, I190T, and G191A FBPases. Structural modeling and kinetic studies suggest that Lys(42), ILe(190), and Gly(191) are located at the pivot point of intersubunit conformational changes that energetically couple the Mg2+-binding site to the AMP domain of FBPas e.