Kinetic of irreversible inactivation of γ-glutamyltranspeptidase from b.subtilis NX-2

γ-Glutamyltranspeptidase (GGT; EC 2.3.2.2) can catalyze the cleavage compounds and the transfer of their γ-glutamyl groups to water or other amino acids and peptides. GGT can be used in the enzymatic synthesis of some glutamyl compounds with special physiological activities, and its inhibition is important for the investigation of its catalytic mechanism. The kinetic theory of the substrate reaction with irreversible inhibition of enzyme activity proposed previously by Tsou was applied for the study of the inactivation of GGT, and the irreversible inhibitor of Woodward's Reagent K (WRK) was used in this study to react with GGT from B. subtilis NX-2. As a result, the irreversible inhibition reaction rate of WRK to GGT,and the combination constant of WRK with enzyme were determined as k_i = 0.03015 s^-1 and KI =1.352 mmol·L^-1, respectively. When the inhibitor presents, the Michaelis-Menten constant and the GGT maximum acylation reaction rate were found as K_m^*= 3.245 mmol·L^-1 and V_max^*= 0.3771 mmol·(L·s)^-1, respectively. Kinetic analysis reveals that the average order of the inactivation reaction is 1.313, and it indicates that there is at least one molecule of Glu/Asp residue exists in the active site of GGT, which plays the role of binding with the substrate during the catalytic reaction.