Site-directed mutagenesis of the conserved histidine residue of phosphoenolpyruvate carboxylase. His138 is essential for the second partial reaction.
Histidine residues have previously been suggested to be essential for the activity of phosphoenolpyruvate carboxylase as demonstrated by chemical modification of these residues. Although the location of these residues on the primary structure is not known, a comparison of nine phosphoenolpyruvate (P-pyruvate) carboxylases sequenced recently revealed that there are only two conserved histidine residues (His138 and His579, coordinates from the E. coli enzyme). Site-directed mutagenesis of these residues were undertaken with the E. coli P-pyruvate carboxylase and the properties of purified mutant enzymes were investigated. Mutation of His138 to asparagine (H138N) produced a protein which did not show carboxylase activity. However, this mutant enzyme catalyzed the bicarbonate-dependent dephosphorylation (Vmax = 1.4 mumol.min-1.mg-1) of the P-pyruvate. Since this reaction is due to one of the two partial reactions proposed for this enzyme, the results indicate that His138 is obligatory for the second-step reaction, i.e. the carboxylation of the enolate form of pyruvate by carboxyphosphate. Mutation of His579 to asparagine (H579N) produced an enzyme which had 69% of the wild-type carboxylase activity, but its affinity for P-pyruvate was decreased by 24-fold.
Terada
K
K
Department of Chemistry, Faculty of Science, Kyoto University, Japan.
Izui
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D005075:Biological Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006639:Histidine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D011994:Recombinant Proteins; D006639:Histidine; D010730:Phosphoenolpyruvate Carboxylase