A bifunctional enzyme, fructose-6-phosphate 2-kinase-fructose 2,6- bisphosphatase, catalyzes synthesis and degradation of fructose 2,6- bisphosphate. Mutants of basic residues, including Lys51, Arg78, Arg79, Arg136, Lys172, and Arg193, immediately around the active site of rat testis fructose 6-P,2-kinase were constructed, and their steady state kinetics, ATP binding, and the effect of pH on the kinetics were characterized. All mutants showed a several-fold increase in K(MgATP), much larger increases in K(Fru 6- P), and decreased V compared to those of the wild type enzyme (WT). Replacement of Lys 172 and Arg 193 with Ala and Leu, respectively, also produced mutants with large K(Fru 6-P) values. Substitution of Lys51, which is located in a Walker-A motif (GXXGXGKT, amino acids 45-52), with Ala or His resulted in enzymes with increased K(MgATP) values and unable to bind Fru 6- P. The dissociation constants for 2'-(3')-O-(N-methylanthraniloyl)-ATP (mantATP) and ATP of all these mutants except Lys51 were similar. Lys51 mutants were unable to bind mantATP. The pH dependence of V and the V/Ks for MgATP and Fru 6-P suggest a mechanism in which reactants and enzyme combine irrespective of the protonation state of groups required for binding and catalysis, but only the correctly protonated enzyme-substrate complex is catalytically active. A chemical mechanism is suggested in which a general base accepts a proton from the 2-hydroxyl of Fru 6-P concomitant with nucleophilic attack on the γ-phosphate of MgATP. Phosphoryl transfer is also facilitated by interaction of the γ-phosphate with a positively charged residue that neutralizes the remaining negative charge. The dianionic form of the 6-phosphate of fructose 6-P is required for binding, and it is likely anchored by a positively charged enzyme residue. A comparison of the pH dependence of kinetic parameters for Ala or His mutant proteins at Lys51, Lys172, and Arg79 suggests that Lys51 interacts with the γ-phosphate of MgATP and that several other arginines likely participate in transition state stabilization of the transferred phosphoryl. The active site general base has yet to be identified.
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