Previous studies of Escherichia coli phosphofructo-1-kinase have shown that mutation of Asp 127 lowers kcat by 5 orders of magnitude. As shown here, introduction of a second mutation (R252Q) that neutralizes a positive charge in the active site increases activity of D127S by 100 fold, suggesting that part of the effect of the Asp mutation may be attributed to non-specific charge interactions. This conclusion is supported by the fact that the R252Q mutant shows a pH dependence that is the reverse of the wild type enzyme, whereas the double mutant has a pH dependence that resembles that of wild type enzyme, although somewhat attenuated.