INACTIVATION AND CONFORMATIONAL-CHANGES OF AMINOACYCLASE IN TRIFLUOROETHANOL SOLUTIONS

The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during d enaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at lo w TFE concentrations. The kinetic theory of the substrate reaction dur ing irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988), Adv. Enzymol. Related Areas Mal. Biol. 61, 381-436 ] was applied to study the kinetics of the inactivation course of amin oacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Ts ou's method. The inactivation reaction was a monophasic first-order re action, The kinetics of the unfolding course were a biphasic process c onsisting of two first-order reactions. Pit 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rat e. At a higher concentration of TFE (10%), the inactivation rate was t oo fast to be determined by conventional methods, whereas the unfoldin g course remained as a biphasic process with fast and slow reactions o ccurring at measurable rates. The results suggest that the aminoacycla se active site containing Zn2+ ions is situated in a limited and flexi ble region of the enzyme molecule that is more fragile to the denatura nt than the protein as a whole.