The fructose 6-phosphate (Fru-6P) saturation curve for phosphofructokinase (PFK) from E. coli is sigmoidal in the presence of saturating MgATP levels, while the corresponding curve for B. stearothermophilus PFK is essentially hyperbolic. Sigmoidality can be due to apparent cooperativity arising from the kinetic mechanism of an enzyme. We have determined the kinetic mechanism of B. stearothermophilus PFK (BsPFK). BsPFK was found to obey a non rapid-equilibrium random mechanism similar to the one E. coli PFK (EcPFK) follows. Substrate inhibition by (...) MgATP was observed. We propose that substrates bind to BsPFK through two alternative pathways, one of which is slower. The substrate inhibition arises in part from reaction flux through the slower pathway. Although EcPFK and BsPFK obey similar kinetic mechanisms, they are inhibited differently by MgATP: EcPFK is profoundly inhibited, BsPFK only weakly. The structural basis for this difference could be closure of the active site via a conformational transition that occurs in EcPFK, but not BsPFK. To investigate the importance of this transition for MgATP inhibition of EcPFK, we have constructed a chimeric enzyme (ChiPFK) that contains the "rigid" ATP-binding domain of BsPFK grafted onto the remainder of the EcPFK subunit. Our results indicate that ChiPFK is locked in an "open" structure resembling the activated form of EcPFK. It is insensitive to heterotropic regulation. Nevertheless, ChiPFK exhibits residual cooperativity. Possible explanations for the cooperativity are discussed. The 6F loop is proposed to be important in PFK allosteric behavior. Residues along the loop are largely conserved between BsPFK and EcPFK, except for 161, which is a glutamate in BsPFK, and a glutamine in EcPFK. Using site-directed mutagenesis, Glu 161 of BsPFK has been changed to glutamine and alanine. Similarly, Gin 161 of EcPFK has been changed to glutamate, arginine and alanine. Of the five mutants, one, QA161, was particularly interesting. Though activated normally by GDP, it was completely insensitive to PEP inhibition. This indicates that the hydrogen-bonding ability of residue 161 is critical for PEP inhibition of EcPFK, and that GDP activation and PEP inhibition follow different structural pathways. (shrink)

Many enzymes are inhibited by their own substrates, leading to velocity curves that rise to a maximum and then descend as the substrate concentration increases. Substrate inhibition is often regarded as a biochemical oddity and experimental annoyance. We show, using several case studies, that substrate inhibition often has important biological functions. In each case we discuss, the biological significance is different. Substrate inhibition of tyrosine hydroxylase results in a steady synthesis of dopamine despite large fluctuations in tyrosine due to meals. (...) Substrate inhibition of acetylcholinesterase enhances the neural signal and allows rapid signal termination. Substrate inhibition of phosphofructokinase ensures that resources are not devoted to manufacturing ATP when it is plentiful. In folate metabolism, substrate inhibition maintains reactions rates in the face of substantial folate deprivation. Substrate inhibition of DNA methyltransferase serves to faithfully copy DNA methylation patterns when cells divide while preventing de novo methylation of methyl‐free promoter regions. (shrink)