title: Influence of a sulfhydryl cross-link across the allosteric-site interface of E. coli phosphofructokinase.
abstract: To assess the role of quaternary stability on the properties of Escherichia coli phosphofructokinase (PFK), a disulfide bond has been introduced across 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-PAGE of oxidized N288C indicates monomeric protein, blocking the six native cysteine residues with N-ethyl maleimide (NEM) reveals dimers of N288C on non-native gels. Subsequent addition of dithiothreitol (DTT) to NEM-labeled N288C regenerates the monomer on SDS-PAGE, reflecting the reversibility of intersubunit disulfide bond formation. KSCN-induced hybrid formation between N288C and the charged-tagged mutant E195,199K exhibits full monomer-monomer exchange only upon DTT addition, providing a novel assessment of disulfide bond formation without NEM treatment. N288C also exhibits a diminished tendency toward nonspecific aggregation under denaturing conditions, a phenomenon associated with monomer formation in PFK. Pressure-induced dissociation and urea denaturation studies further indicate that oxidized N288C exhibits increased quaternary stability along both interfaces of the tetramer, suggesting a synergistic relationship between active site and allosteric site formation. Although the apparent binding affinities of substrates and effectors change somewhat upon disulfide formation in N288C, little difference is evident between the maximally inhibited and activated forms of the enzyme in oxidizing versus reducing conditions. Allosteric influence, therefore, is not correlated to subunit-subunit affinity, and does not involve substantial interfacial rearrangement.
authors.affiliation: Department of Chemistry, Southwestern Oklahoma State University, Weatherford, Oklahoma 73096, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Johnson
authors.forename: M D
authors.initials: MD
authors.lastname: Lasagna
authors.forename: G D
authors.initials: GD
authors.lastname: Reinhart
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000737:chemistry*; D011312:Pressure; D017510:Protein Folding; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014508:Urea
chemical_list: D004220:Disulfides; D013439:Sulfhydryl Reagents; D014508:Urea; D010732:Phosphofructokinase-1; D004229:Dithiothreitol