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

Hiroyuki Mizuguchi, Paul F. Cook, Charles A. Hasemann, Kosaku Uyeda

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)8775-8784
Number of pages10
JournalBiochemistry
Volume36
Issue number29
DOIs
StatePublished - Jul 22 1997

ASJC Scopus subject areas

  • Biochemistry

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