EFFECT OF SUBUNIT DISSOCIATION, DENATURATION, AGGREGATION, COAGULATION, AND DECOMPOSITION ON ENZYME INACTIVATION KINETICS .2. BIPHASIC AND GRACE PERIOD BEHAVIOR

A model previously developed to characterize enzymatic inactivation behavior was used to explain the non-first-order biphasic and grace period phenomena that are often observed with oligomeric enzymes. Luciferase and urease were used as model enzymes for this study. It was hypothesized that with a dimeric enzyme such as luciferase, the oligomer initially dissociates reversibly into two native monomer species. These native monomers can then reversibly denature and irreversibly aggregate and coagulate. With the hexamer, urease, two trimers are formed that can subsequently aggregate to form an inactive hexamer. The dissociated monomer species of luciferase do not possess catalytic activity, so the inactivation mechanism is biphasic; the first slope of a-first-order kinetic plot is influenced by the reversible oligomer/monomer/denatured-monomer transition whereas the second slope is associated with either irreversible aggregation or coagulation. In contrast, the trimer of urease has the same activity as the hexamer; therefore, during the initial hexamer-trimer transition, little activity loss occurs. However, as the trimer concentration increases, activity decreases as a result of trimer aggregation. As a result, grace period inactivation behavior is observed.