Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.
Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.
Berger
S A
SA
MRC Laboratory of Molecular Biology, Cambridge, England.
Evans
P R
PR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006153:Guanosine Diphosphate / Q000494:pharmacology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1