Phosphofructo-1-kinase: role of charge neutralization in the active site.
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.
Zheng
R L
RL
Department of Biological Chemistry, Chicago Medical School North Chicago, IL 60064, USA.
Kemp
R G
RG
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase