Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase

Abstract

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.

The cooperativity and allosteric inhibition of Escherichia coli phosphofructokinase depend on the interaction between threonine-125 and ATP.

“…It is more difficult to predict the effect of a mutation of Arg-72 since this residue is engaged in an ionic bond in both the R and T states, albeit with different partners. Like all changes around the y-phosphate of ATP (7,13), the Arg-72 --Ser mutation decreases the cooperativity of Fru-6P saturation (31). The Arg-72 -* Ser mutant is less inhibited by PEP than wild type (Fig.…”

“…The side chain of Thr-125 does not significantly contribute to the affinity for ATP; for the Thr-125 -* Ala (13) and Thr-125 -* Ser (Table 2) mutants, Km for ATP is, respectively, 1.5 and 3 times lower than that for wild-type PFK. This result is unexpected since x-ray crystallography suggests that the hydroxyl group of Thr-125 interacts with the -y-phosphate of (o) as substrate.…”

“…In the x-ray structure of the R state of PFK with substrates and/or products bound, the phosphoryl group transferred from ATP to Fru-6P interacts with (5,7,13 *To whom reprint requests should be addressed.…”

“…PFK activity was measured using a coupled assay (16) a slight perturbation of the OH group, whereas the Thr-125 -) Ala mutation, which removes this group, decreases kt by three orders of magnitude (13). Therefore the value of kcat depends on the presence and correct positioning of the hydroxyl group.…”

“…In both cases the ordinate represents the fractional saturation relative to maximum velocities, which are, respectively, 2.5 times (Table 2) and 400 times ( (5,7). This hydrogen bond remains to be characterized by x-ray crystallography for the Thr-125 -+ Ser mutant, but if no hydrogen bond was present in this mutant, its kcat would be expected to decrease by 1000-fold, like that of the Thr-125 -> Ala mutant (13), and not by only 2.5-fold (Table 2). …”