Rabbit Muscle Phosphofructokinase

Crystalline phosphofructokinase from rabbit skeletal muscle contains 16 to 18 thiol groups per protomer (93,200 daltons). Six groups reacted immediately with 5,5'-dithiobis-(2-nitrobenzoic acid). Pseudo-first order rate constants of 0.0265, 0.0138, and 0.0082 per min were obtained for the seventh plus eighth, ninth plus 10th, and 11th plus 12th thiol groups, respectively, in 0.025 m glycylglycine buffer, pH 8.0, at 28°. The remaining four to six groups reacted very slowly. All thiol groups reacted with Ellman's reagent in 6 m urea, 4 m guanidine hydrochloride, or 3 mm sodium dodecyl sulfate. The thiol content determined by Ellman's reagent approached the half-cystine content determined by amino acid analysis. In the absence of a denaturing agent, fructose 6-phosphate protected two of the six rapidly reacting thiol groups. In the presence of 4 m urea, fructose 6-phosphate as well as fructose 1,6-diphosphate diminished the rate of reaction of the thiol groups with Ellman's reagent. Fructose 6-phosphate seemed to protect highly reactive groups, whereas fructose 1,6-diphosphate exerted its influence on slowly reacting ones. Phosphofructokinase activity was sensitive to 1 mm iodoacetamide. The substrates fructose 6-phosphate and MgATP offered complete protection against this inhibition while the product fructose 1,6-diphosphate did not. About 60% of the enzymatic activity was lost upon alkylation of six thiol groups per protomer.