Catalytic role of an arginine residue in the highly conserved and unique sequence of phosphoenolpyruvate carboxylase.
Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] has a highly conserved and unique sequence, 578-FHGRGGSIGRGGAP-591 (on Escherichia coli, PEPC), in which a GRGG motif is repeated twice with two intervening residues. Since previous chemical modification studies suggested the functional importance of arginine residues, the invariant Arg587 in this region was replaced with Ser, and the enzymatic properties of the resulting mutant enzyme (R587S) were investigated. Replacement led to virtual loss of the catalytic activity to form oxaloacetate. The specific activity was 37 nmol.min-1.mg-1, which corresponds to 2 x 10(-4)-fold the activity of the wild-type enzyme. However, the activity of bicarbonate- and Mg(2+)-dependent hydrolysis of phosphoenolpyruvate (PEP) to pyruvate appeared for the mutant enzyme with a specific activity of 2.1 mumol.min-1.mg-1. In view of the stepwise reaction mechanism proposed for PEPC, this activity can be attributed to impairment of the subsequent partial reaction(s) following the formation of the intermediate carboxyphosphate. The half-saturation concentration (S0.5) of HCO3- in R587S was about 100-fold that in the wild-type enzyme, whereas the respective values for PEP and Mg2+ were 20- and 15-fold, indicative of this residue participating in the binding of HCO3-.
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D001639:Bicarbonates / Q000378:metabolism; D002384:Catalysis; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment
D001639:Bicarbonates; D018698:Glutamic Acid; D010728:Phosphoenolpyruvate; D001120:Arginine; D010730:Phosphoenolpyruvate Carboxylase; D008274:Magnesium