Role of residue 161 in the allosteric transitions of two bacterial phosphofructokinases. 
The loop between alpha-helix 6 and beta-strand F of the phosphofructokinase (PFK) from Bacillus stearothermophilus is proposed to be important in the allosteric transition of the enzyme [Schirmer, T., & Evans, P.R. (1990) Nature 343, 140-145].  Except for residue 161, the amino acids within the loop are similar between B. stearothermophilus PFK (BsPFK) and the PFK from Escherichia coli (EcPFK).  In the former enzyme, residue 161 is a glutamate, while in the latter it is a glutamine.  We have used site-directed mutagenesis to investigate the importance of residue 161 for the allosteric regulation of the two enzymes by phosphoenolpyruvate (PEP), an inhibitor, and GDP, an activator.  In BsPFK, glutamate 161 has been changed to a glutamine and an alanine, while in EcPFK, glutamine 161 has been changed to a glutamate, an arginine, and an alanine.  The kinetic parameters of the mutant enzymes were similar to those of the respective wild types, indicating that residue 161 is not directly involved in substrate binding and catalysis.  One of the EcPFK mutants, Q161A, though activated normally by GDP, was completely insensitive to PEP.  This indicates that the hydrogen-bonding ability of residue 161 is critical for PEP inhibition of EcPFK and suggests that GDP activation and PEP inhibition follow different structural pathways in EcPFK.  The BsPFK mutant enzymes were less sensitive to PEP inhibition and more sensitive to GDP activation, suggesting that inhibition and activation are opposed and follow a common structural pathway in agreement with a concerted allosteric mechanism.