title: Designing an allosterically locked phosphofructokinase.
abstract: Six site-directed mutants of Escherichia coli phosphofructokinase (PFK) were made in an attempt to produce an enzyme "locked" in the inactive or "T"-state. The kinetic properties of the mutants were examined as a function of the substrates fructose 6-phosphate (Fru6P) and ATP, the positive effector GDP, and the negative effector phosphoenolpyruvate (PEP). All mutants exhibited lower activity than wild-type PFK. Three mutants (RS63, LV153, and VT246) had apparent dissociation constants for substrates and effectors similar to those of wild type. One mutant, HN160, had a 10-fold reduced affinity for Fru6P and reduced apparent affinity for the effectors. Two mutants, SN159 and T(GS)156, exhibited hyperbolic kinetics consistent with a "locked" T-state protein. Surprisingly, T(GS)156 showed hyperbolic activation in response to the physiological inhibitor PEP. The mutant PFK properties are discussed in terms of the PFK structure. These results suggest that the kinetic properties of PFK are sensitive to interactions in the homotropic interface; residues 156-160 in particular are critical in mediating the interactions between effector and active sites and in the T to R quaternary transition.
authors.affiliation: Laboratory of Molecular Biology, Medical Research Council, Cambridge, U.K.
authors.forename: C E
authors.initials: CE
authors.lastname: Kundrot
authors.forename: P R
authors.initials: PR
authors.lastname: Evans
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D015345:Oligonucleotide Probes; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1