title: Active-site mutants altering the cooperativity of E. coli phosphofructokinase.
abstract: Crystal structures of the high- and low-activity states of the allosteric enzyme phosphofructokinase implicate three arginines in substrate binding, catalysis and cooperativity. Arginines 162 and 243 reach into the active site from an adjacent subunit and interact with the cooperative substrate fructose 6-phosphate. Mutation of these arginines to serine results in mutant enzymes with reduced substrate binding and lowered cooperativity, but with little change in their catalytic ability (kcat). Arg 72 bridges the two substrates fructose 6-phosphate and ATP, and interacts with the 1-phosphate of the product fructose 1,6-biphosphate. Mutation of this residue to serine reduces the catalytic activity, cooperativity and binding of fructose 6-phosphate and fructose 1,6-bisphosphate. In the reverse reaction, the kinetics of wild-type and the Ser 72 mutant with respect to fructose 1,6-bisphosphate are hyperbolic, whereas those of the Ser 162 and Ser 243 mutants are sigmoidal. These results show that each of the three arginines contributes to cooperativity and to the transmission of allosteric signals between the four subunit of the enzyme.
authors: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors: S A
authors: SA
authors: Berger
authors: P R
authors: PR
authors: Evans
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001120:Arginine; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005636:Fructosephosphates / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000494:pharmacology; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D005636:Fructosephosphates; D006153:Guanosine Diphosphate; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D010732:Phosphofructokinase-1