Fluorescence Studies of the Binding of Alkyl and Aryl Phosphates to Rat Muscle Aldolase

A series of aromatic phosphates have been prepared and tested as inhibitors of fructose 1,6-diphosphate cleavage by rat muscle aldolase (EC 4.1.2.b). Naphthalenediol diphosphates have dissociation constants (Ki) ranging from 2.2 x 10-6 to 3.5 x 10-7 m, depending on the relative positions of the phosphate groups. Phenyl phosphatate (Ki = 3.1 x 10-3 m), 1-naphthol phosphate (Ki = 5.3 x 10-4 m), and resorcinol diphosphate (Ki = 8.8 x 10-7 m), have also been examined. Equilibrium dialysis and spectrophotometric and fluorimetric titration demonstrate that the binding is specific and that there are 3.3 ± 0.2 sites per mole of aldolase. The fluorescence of some naphthol diphosphates is enhanced on binding, suggesting that the binding sites of the enzyme provide a hydrophobic environment. The quenching of aldolase fluorescence by a number of aliphatic mono- and diphosphates indicates that tryptophan may be present in or close to the binding sites. A rapid and convenient procedure for the isolation of crystalline rat muscle aldolase by ammonium sulfate fractionation is described.