Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. Mutation at the substrate-binding site affects allosteric behavior.
Arg252 of fructose-6-phosphate 1-kinase (PFK) from Bacillus stearothermophilus has been proposed to be involved in the binding of the substrate Fru-6-P. We demonstrate here that mutation of this residue to alanine converts the enzyme to a form with characteristics similar to those of its allosterically tight form. The mutant enzyme exhibits a high affinity for its inhibitor phosphoenolpyruvate (a 68-fold difference compared to wild type) and a dramatically decreased Fru-6-P affinity (1500-fold increase in Km). It is more sensitive to inhibition by high ATP concentrations than the wild type, and this inhibition is relieved by ADP, GDP, or higher Fru-6-P concentrations. In contrast, mutation of Arg252 to lysine increases the affinity of the enzyme for P-enolpyruvate by only 2-fold and increases its Km for Fru-6-P by only 50-fold. Sigmoidal kinetics with respect to Fru-6-P in the presence of P-enolpyruvate were observed with Hill numbers of 2.2, 2.4, and 1.7 for wild-type B. stearothermophilus PFK and the Arg252 to lysine and to alanine mutations, respectively. Unlike fructose-6-phosphate 1-kinase from Escherichia coli, in the absence of P-enolpyruvate, B. stearothermophilus PFK exhibits a hyperbolic profile with respect to Fru-6-P concentration. B. stearothermophilus PFK is sensitive to inhibition by high ATP concentrations and competitively inhibited by GDP or ADP. Our data indicate that Arg252 of B. stearothermophilus PFK plays a major role in both Fru-6-P binding and allosteric interaction between the subunits. However, this residue does not seem to participate directly in the catalytic process.
Valdez
B C
BC
Department of Biochemistry, Louisiana State University, Baton Rouge 70803.
French
B A
BA
Younathan
E S
ES
Chang
S H
SH
D000494:Allosteric Regulation; D001120:Arginine; D001665:Binding Sites; D003001:Cloning, Molecular; D005796:Genes; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*
D001120:Arginine; D010732:Phosphofructokinase-1