Studies of 6-Phosphogluconate Dehydrogenase from Sheep Liver. 2. Kinetics of the Oxidative-Decarboxylation Reaction, Coenzyme Binding and Analyses for Metals

1 6-Phosphogluconate dehydrogenase purified from sheep liver does not require added bivalent metal ions for activity, and appears not to contain significant amounts of firmly bound Mn2+, Mg2+ or Zn2+. 2 Kinetic studies of the oxidative decarboxylation reaction have been made in phosphate buffer pH 7.0 and pH 8.0, in triethanolamine buffer pH 7.0 and in phosphate buffer pH 7.0 in the presence of Mg2+, which activates the enzyme. The initial-rate parameters are recorded, and vary significantly with pH and the nature of the buffer. 3 Fluorescence titrations of the enzyme with NADPH indicate two binding sites per enzyme molecule, and the dissociation constant in phosphate buffer is very approximately 0.2 μM. 4 The data are consistent with a compulsory order mechanism in which, at pH 7.0 in triethanolamine buffer, ternary complexes are not kinetically significant.