Site-directed mutagenesis of phosphoenolpyruvate carboxylase from E. coli: the role of His579 in the catalytic and regulatory functions.
Phosphoenolpyruvate carboxylases (PEPC) [EC 4.1.1.31] from a wide variety of organisms contain a unique and highly conserved sequence, 578FHGRGGSIGRGGAP591 (coordinates for the Escherichia coli enzyme), which has been presumed to participate in the binding of phosphoenolpyruvate (PEP). Since previous chemical modification studies had suggested the importance of His for the catalytic activity, the role of His579 was investigated by constructing variants of E. coli PEPC, in which this residue was substituted to Asn (H579N) or Pro (H579P). Kinetic studies with partially purified enzymes revealed the following: (1) The apparent maximal velocities in the presence of acetyl-CoA (CoASAc, one of the allosteric activators) were 29% and 5.4% of the wild-type enzyme, for H579N and H579P, respectively. (2) The half-saturation concentration for PEP was increased about 40-fold by the substitutions, while those for another substrate (HCO3-) and the metal cofactor (Mg2+) were increased only 2- to 4-fold. (3) The half-saturation concentrations of four kinds of allosteric activators and of dioxane, an artificial activator, were also changed to various extents. Among them the most remarkable increase was observed for CoASAc (28-fold). (4) The concentration of an allosteric inhibitor, aspartate, required for 50% inhibition remained substantially unchanged. It was concluded that the imidazole group of His579 is not obligatory for the enzyme catalysis, but plays important roles in catalytic and regulatory functions.
Terada
K
K
Department of Chemistry, Faculty of Science, Kyoto University.
Murata
T
T
Izui
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D006639:Histidine; D010730:Phosphoenolpyruvate Carboxylase