Spin-labelled phosphofructokinase. A simple and direct approach to the study of allosteric equilibria under near-physiological conditions.
Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non-exclusive binding of effectors. On this basis MgATP, fructose 6-phosphate plus ATP, and NH+4ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6-phosphate and fructose 1,6-bisphosphate are less potent activators, and free ATP and H+ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups. Strong positive cooperativity occurs under suitable conditions with ATP, metal-ATP and fructose 6-phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6-phosphate because of a distinct concerted effect on conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple models; phosphates favour the binding, and reduce the cooperativity, of fructose 6-phosphate and metal-ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6-phosphate. The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation.
D000255:Adenosine Triphosphate; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D002951:Citrates; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008274:Magnesium; D009132:Muscles / Q000201:enzymology; D010732:Phosphofructokinase-1; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis
D002951:Citrates; D008024:Ligands; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D008274:Magnesium