The Influence of Ligands on the Fluorescence of the Complex between 8-Anilino-1-naphthalene Sulfonate and Pyruvate Kinase of Neurospora

Abstract

The fluorescence of 8-anilino-1-naphthalene sulfonate (ANS) bound to pyruvate kinase (PK) was diminished by the allosteric effector fructose 1,6-diphosphate (FDP). One of the substrates, phosphoenolpyruvate (PEP), stabilized the binding of FDP to the enzyme. The fluorescence of ANS and its reduction by FDP was studied as a function of temperature and pH: at low temperatures (12–15 °C) the relative quantum yields of the bound ANS were high but virtually no binding of FDP occurred. The optimum temperature and pH for FDP binding was observed to lie in the range 22–26 °C and 7.0–8.0, respectively. On incubation of PK with ANS at 22 °C and 35 °C under conditions corresponding to those employed for fluorescence studies, no inactivation of the enzyme was encountered; incubation at 3–4 °C led to a loss of 85–90% of the activity in 60 min. PEP and FDP, but not ADP, protected the enzyme against ANS-induced inactivation. In contrast, inactivation experiments conducted with rabbit muscle PK showed that both PEP and ADP afforded protection but FDP was completely ineffective.