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To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1±C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited k cat , K m and I 50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg 21 was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had . 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1±C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.Keywords: AMP inhibition; cooperativity; FBPase; fructose-1,6-bisphosphatase; site-directed mutagenesis.Fructose-1,6-bisphosphatase (d-fructose-1,6-bisphosphate 1-phosphohydrolase; EC 3.1.3.11; FBPase) catalyzes the hydrolysis of fructose 1,6-bisphosphate (Fru1,6P 2 ) to fructose 6-phosphate (Fru6P) and inorganic phosphate, a rate limitingreaction of renal and hepatic gluconeogenesis [1,2]. The pigkidney enzyme is a homotetramer with a molecular mass of 146 000 Da [3,4]. Its activity is regulated physiologically by the inhibitors fructose 2,6-bisphosphate (Fru2,6P 2 ) and AMP [1,5,6]. AMP binds noncompetitively to an allosteric site [6±8], whereas Fru2,6P 2 binds to the active sites of the enzyme in competition with the substrate Fru1,6P 2 [7,9,10]. In addition, AMP and Fru2,6P 2 exhibit synergism in their inhibitions [10,11]. The enzyme requires bivalent metal ions such as Mn 21 , Mg 21 or Zn 21 to achieve catalytic activity [1,2]. The magnesium saturation and AMP inhibition are cooperative processes with Hill coefficients between 2.0 and 2.5 [6,12]. AMP and Mg 21 are mutually exclusive in their binding to the enzyme, indicating that this could be the mechanism for the AMP regulation of the FBPase [13,14].X-ray crystallographic studies have shown that FBPase is a tetrameric molecule with four identical polypeptide chains that aggregate into a flat hexagonal disk with D 2 symmetry [8,15]. The tetramer has the upper left vertex occupied by subunit C1 followed by C2, C3 and C4. The four subunits of FBPase are designated C1, C2, C3 and C4 and are labeled clockwise. The C1 and C2 subu...

The expression of aldolase A and B isoenzyme transcripts was confirmed by RT-PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose-1,6-bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co-localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 microM Fru-1,6-P2. These data indicate the presence of a modulated enzyme-enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis.

The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving as the first intermediate a catalytically inactive tetramer. The evidence that indicates the existence of this intermediate is as follows: (1) the loss of enzymatic activity and the concomitant increase of ANS binding, at low concentrations of Gdn.HCl (midpoint at 0.75 M), are both protein concentration independent, and (2) the enzyme remains in a tetrameric state at 0.9 M Gdn.HCl as shown by size-exclusion chromatography. At slightly higher Gdn.HCl concentrations the inactive tetramer dissociates to a compact dimer which is prone to aggregate. Further evidence for dissociation of tetramers to dimers and of dimers to monomers comes from the concentration dependence of AEDANS-labeled enzyme anisotropy data. Above 2.3 M Gdn.HCl the change of AEDANS anisotropy is concentration independent, indicative of monomer unfolding, which also is detected by a red shift of MIANS-labeled enzyme emission. At Gdn.HCl concentrations higher than 3.0 M, the protein elutes from the size-exclusion column as a single peak, with a retention volume smaller than that of the native protein, corresponding to the completely unfolded monomer. In the presence of its cofactor Mg(2+), the denaturated enzyme could be successfully reconstituted into the active enzyme with a yield of approximately 70-90%. Refolding kinetic data indicate that rapid refolding and reassociation of the monomers into a nativelike tetramer and reactivation of the tetramer are sequential events, the latter involving slow and small conformational rearrangements in the refolded enzyme.

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