CHEMICAL MECHANISM OF THE FRUCTOSE-6-PHOSPHATE,2-KINASE REACTION FROMTHE PH-DEPENDENCE OF KINETIC-PARAMETERS OF SITE-DIRECTED MUTANTS OF ACTIVE-SITE BASIC RESIDUES

A bifunctional enzyme, fructose-6-phosphate 2-kinase-fructose 2,6-bisp hosphatase, catalyzes synthesis and degradation of fructose 2,6-bispho sphate. Mutants of basic residues, including Lys51, Arg78, Arg79, Arg1 36, Lys172, and Arg193, immediately around the active site of rat test is fructose 6-P,2-kinase were constructed, and their steady state kine tics, ATP binding, and the effect of pH on the kinetics were character ized, All mutants showed a several-fold increase in K-MgATP, much larg er increases in KFru6-P, and decreased V compared to those of the wild type enzyme (WT). Replacement of Lys172 and Arg193 with Ala and Leu, respectively, also produced mutants with large KFru6-P values. Substit ution 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-MgA TP values and unable to bind Fru6-P, The dissociation constants for 2' -(3')-O-(N-methylanthraniloyl)-ATP (mantATP) and ATP of all these muta nts except Lys51 were similar, Lys51 mutants were unable to bind mantA TP, The pH dependence of V and the V/Ks for MgATP and Fru6-P suggest a mechanism in which reactants and enzyme combine irrespective of the p rotonation state of groups required for binding and catalysis, but onl y the correctly protonated enzyme-substrate complex is catalytically a ctive. A chemical mechanism is suggested in which a general base accep ts a proton from the 2-hydroxyl of Fru6-P concomitant with nucleophili c attack on the gamma-phosphate of MgATP, Phosphoryl transfer is also facilitated by interaction of the gamma-phosphate with a positively ch arged residue that neutralizes the remaining negative charge. The dian ionic 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 c omparison of the pH dependence of kinetic parameters for Ala or His mu tant proteins at Lys51, Lys172, and Arg79 suggests that Lys51 interact s with the gamma-phosphate of MgATP and that several other arginines l ikely participate in transition state stabilization of the transferred phosphoryl, The active site general base has yet to be identified.