Proteolysis of Immobilized Lactate Dehydrogenase in the Presence of Pyruvate and NADH
We have described the use of enzyme immobilization in a flow system as a means to detect substrate induced conformational changes of enzymes (1,2). The enzyme and substrate were mixed just prior to contacting a column of activated solid support. The enzyme forms bound in the presence and absence of substrate were then studied in chemical modification and kinetic experiments. In our new approach we study the susceptibility of a bound enzyme to proteolysis in the presence and absence of substrates. Susceptibility to proteolysis is a useful property in the study of conformational changes of proteins (3). However, the conformation of the ES complex has not been studied because of the difficulty in maintaining saturating levels of substrate.