Abstract
The significance of interactions between AMP domains in recombinant porcine fructose-1,6-bisphosphatase (FBPase) is explored by site-directed mutagenesis and kinetic characterization of homogeneous preparations of mutant enzymes. Mutations of Lys42, Ile190, and Gly191 do not perturb the circular dichroism spectra, but have significant effects on ligand binding and mechanisms of cooperativity. The Km for fructose 1,6-bisphosphate and the Ki for the competitive inhibitor, fructose 2,6-bisphosphate, decreased by as much as 4- and 8-fold, respectively, in the Q32L, K42E, K42T, I190T, and G191A mutants relative to the wild-type enzyme. Q32L, unlike the other four mutants, exhibited a 1.7-fold increase in Kcat. Mg2+ binding is sigmoidal for the five mutants as well as for the wild-type enzyme, but the Mg2+ affinities were decreased (3-22-fold) in mutant FBPases. With the exception of Q32L (8-fold increase), the 50% inhibiting concentrations of AMP for K42E, K42T, I190T, and G191A were increased over 2,000-fold (>10 mM) relative to the wild-type enzyme. Most importantly, a loss of AMP cooperativity was found with K42E, K42T, I190T, and G191A. In addition, the mechanism of AMP inhibition with respect to Mg2+ was changed from competitiv...Continue Reading
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