Resolution of the pair-wise allosteric interactions found in phosphofructokinase from Bacillus stearothermophilus

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a homotetrameric enzyme with an average of one active site and one allosteric site per subunit. BsPFK is inhibited by phosphoenolpyruvate (PEP) and how this inhibitory signal is propagated throughout the enzyme is the main question we address through this investigation. By possessing a total of eight binding sites, a potential for twenty-eight total pair-wise allosteric interactions result within BsPFK, ten of which are unique. Of these ten interactions, four are heterotropic interactions, or interactions between unlike binding sites, while the remaining six interactions are homotropic interactions, or interactions between like binding sites. Thus, to address the question of how BsPFK is inhibited by PEP, each of these ten interactions needs to be quantified and their roles in the inhibition process assessed. In order to quantify the roles of the 10 allosteric interactions, we created, purified and characterized several different hybrid enzymes by using site-directed mutagenesis to reduce the number of native active sites and native allosteric sites to permit the isolation of specific allosteric interaction(s). Through the creation and isolation of 1:3 hybrid enzymes, in which one native active site and one native allosteric site remain, each of the four heterotropic interactions were characterized. Moreover, through the creation and isolation of the 2:2 hybrid enzymes, in which two native active sites and two native allosteric sites remain, characterization of the remaining six homotropic interactions was performed. Utilizing a linked function approach to quantify the heterotropic and homotropic effects for each hybrid enzyme, we determined that 5 to 6 of the ten pair-wise allosteric interactions found in BsPFK are involved in the inhibition process depending upon pH. More importantly however, our data provides definitive results that the traditional two-state models used to describe an allosteric effect are not sufficient to describe the allosteric effect measured for BsPFK. Rather, our results show that the linked function approach is a more appropriate way to unambiguously measure the nature and magnitude of an allosteric effect. Moreover, this approach can also be used to explain the allosteric behavior of a dimeric enzyme.