Chemistry of Enzymatic ATP Synthesis: An Insight through the Isotope Window

A study was conducted to demonstrate that an isotope provided significant information about chemistry of enzymatic ATP synthesis. Isotope-containing MgCl 2, CaCl 2, and ZnCl 2 samples were obtained using the treatment of magnesium, calcium, and zinc oxides with analytically pure HCl. Monomeric creatine kinase was purified from Vipera xanthia venom, phosphoglycerate kinase was purified from pig skeletal muscle, and pyruvate kinase was isolated and purified from rabbit muscles. The catalytic activities of enzymes were measured conventionally as the amounts of labeled ATP formed in the presence of [ 32P]phosphocreatine, [ 32P] phosphoglycerate, or [ 32P]phosphopyruvate in 1minute at optimal incubation conditions and corrected to 1 mg of tested pure enzyme. The original electrophoretic technique was employed for loading or substitution of magnesium, calcium, and zinc ions into the enzyme active sites.