KINETICS AND HEAT-INACTIVATION MECHANISMS OF D-AMINO-ACID OXIDASE

A study was made of the stability of crude hog kidney D-amino acid oxi dase (D-AAO) under different experimental conditions of temperature, e nzyme concentration, buffer and in the presence of flavine adenine din ucleotide (FAD) and glycerol. A deactivation mechanism is proposed. Th e kinetic deactivation studies were performed in a buffer at pH 8 over a temperature range of 277-327 K (4-54 degrees C). The activity for D -AAO at 1 U/ml at 277 K in potassium phosphate and potassium pyrophosp hate buffer remains almost constant for 20 days. For D-AAO at 0.02 U/m l at 303 K (30 degrees C) in phosphate buffer, the presence of FAD and glycerol causes the deactivation constant to decrease, while the init ial activity and the half-life time increase, the latter doubting on p assing from buffer alone to buffer with FAD and 10% glycerol. In the c ase of D-AAO at 0.2 U/ml, the stability increases considerably with re spect to 0.02 U/ml. Both FAD and glycerol enhance this stabilization. For temperatures above 313 K (40 degrees C), the deactivation of D-AAO at 0.2 U/ml showed values of the thermodynamic variables for the over all deactivation constant and for the deactivation constant due to the loss of FAD that indicate that the dissociation of FAD is the main de activation mechanism. Finally, deactivation due to the loss of FAD is higher for D-AAO at 0.1 U/ml as compared with a concentration of 0.2 U /ml.