Studies on Heart Phosphofructokinase

A procedure has been described for photo-oxidation of crystalline sheep heart phosphofructokinase in the presence of methylene blue. The enzyme after photo-oxidation was insensitive to ATP inhibition. Kinetic studies at pH 8.2 before and after photo-oxidation of the enzyme revealed no significant change in the Km for either fructose 6-phosphate or ATP. Kinetic studies at pH 6.9 revealed a decrease in both the Km for ATP and the Km for fructose-6-P in the photo-oxidized enzyme. The Hill coefficient of the enzyme was 3.15 before photo-oxidation and was decreased to 1.1 after photo-oxidation, indicating disappearance of homotropic interactions between different fructose-6-P sites. The photo-oxidized enzyme was not inhibited by citrate nor activated by AMP. The stability of the enzyme at pH 6.5 was increased after photo-oxidation. ATP protected the enzyme against inactivation during photo-oxidation at pH 8.0 but not at pH 6.9. Fructose-6-P, on the other hand, protected the enzyme at both hydrogen ion concentrations. Neither of the two substrates prevented the desensitization of the enzyme to ATP inhibition following photo-oxidation. The results indicate that desensitization of the enzyme to ATP inhibition is related to the presence of the enzyme in a stable conformation.