Influence of a sulfhydryl cross-link across the allosteric-site interface of E-coli phosphofructokinase

To assess the role of quaternary stability on the properties of Escherichia coli phosphofructokinase (PFK). a disulfide bond has been introduced acros s the subunit interface containing the allosteric binding sites in E. coli phosphofructokinase by changing N288 to cysteine. N288 is located in close proximity to the equivalent residue on an adjacent subunit. Although SDS-PA GE of oxidized N288C indicates monomeric protein, blocking the six native c ysteine residues with N-ethyl maleimide (NEM) reveals dimers of N288C on no n-native gels. Subsequent addition of dithiothreitol (DTT) to NEM-labeled N 288C regenerates the monomer on SDS-PAGE. reflecting the reversibility of i ntersubunit disulfide bond formation. KSCN-induced hybrid formation between N288C and the charged-tagged mutant E195.199K exhibits full monomer-monome r exchange only upon DTT addition, providing a novel assessment of disulfid e bond formation without NEM treatment. N288C also exhibits a diminished te ndency toward nonspecific aggregation under denaturing conditions, a phenom enon associated with monomer formation in PFK. Pressure-induced dissociatio n and urea denaturation studies further indicate that oxidized N288C exhibi ts increased quaternary stability along both interfaces of the tetramer. su ggesting a synergistic relationship between active site and allosteric site formation. Although the apparent binding affinities of substrates and effe ctors change somewhat upon disulfide formation in N288C. little difference is evident between the maximally inhibited and activated forms of the enzym e in oxidizing versus reducing conditions. Allosteric influence. therefore, is not correlated to subunit-subunit affinity, and does not involve substa ntial interfacial rearrangement.