Chemical modification of the most reactive thiol group of rabbit skeletal muscle phosphofructokinase, to reduce its affinity toward substrate ATP and activating monovalent cations

The most reactive single thiol group of rabbit skeletal muscle phosphofructokinase per protomer was modified with the following thiol reagents: iodoacetamide, iodoacetate, 2-hydroxyethyl disulfide, 3,3-dithiodipropionate, and glutathione disulfide. As a result of the modification, there was increase in not only the apparent activation constants of activating monovalent cations, NH,+ (about 3-, 9-, 12-, 20-, and 30-fold, respectively) and K+ (about 3-, 10-, 15-, 17-, and 20-fold, respectively), but also the apparent K ^ for ATP (about 3-, 10-, 15-, 100-, and 20-fold, respectively) without any significant change in maximum velocity or apparent Km for fructose 6-phosphate in the presence of high concentrations of NH,+. These results suggest that modification of the thiol group destabi-lizes the enzyme-monovalent cation-MgATP complex proposed by Suelter [Science (1970) 168, 789-795], causing an apparent loss in catalytic activity. Phosphofructokinase (PFK) (ATP: D-fructose-6-phosphate l-phosphotransferase, EC 2.7.1.11) is a key regulatory enzyme in glycolysis (1). Rabbit skeletal muscle PFK possesses a single thiol group having very high reactivit