Chemical mechanism of the fructose-6-phosphate,2-kinase reaction from the 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-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 KMgATP, much larger increases in KFru 6-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 KFru 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 KMgATP 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 gamma-phosphate of MgATP. Phosphoryl transfer is also facilitated by interaction of the gamma-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 gamma-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.
Mizuguchi
H
H
Research Service, Department of Veterans' Affairs Medical Center, Dallas, Texas 75216, USA.
Cook
P F
PF
Hasemann
C A
CA
Uyeda
K
K
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D025481:Phosphofructokinase-2; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013737:Testis / Q000201:enzymology; D062367:ortho-Aminobenzoates / Q000378:metabolism
D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2