Kinetics and Heat-inactivation mechanisms of d-amino acid oxidase

A study was made of the stability of crude hog kidney d -amino acid oxidase ( d -AAO) under different experimental conditions of temperature, enzyme concentration, buffer, and in the presence of flavine adenine dinucleotide (FAD)_ and glycerol. A deactivation mechanism is proposed. The kinetic deactivation studies were performed in a buffer at pH over a temperature range of 277-327K (4–54°C). The activity for d -AAO at 1 U/ml at 277 K in potassium phosphate and potassium pyrophosphate buffer remains almost constant for 20 days. For d -AAO at 0·02 U/ml at 303 K (30°C) in phosphate buffer, the presence of FAD and glycerol causes the deactivation constant to decrease, while the initial activity and the half-life time increase, the latter doubling on passing from buffer alone to buffer with FAD and 10% glycerol. In the case of d -AAO at 0·2 U/ml, the stability increases considerably with respect to 0·02 U/ml. Both FAD and glycerol enhance this stabilization. For temperatures above 313 K (40°C), the deactivation of d -AAO at 0·2 U/ml showed values of the thermodynamic variables for the overall deactivation constant and for the deactivation constant due to the loss of FAD that indicate that the dissociation of FAD is the main deactivation 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.