Inhibitory kinetics of bromacetic acid on beta-N-acetyl-D-glucosaminidase from prawn (Penaeus vannamei).

beta-N-Acetyl-D-glucosaminidase (NAGase, EC.3.2.1.52), a composition of chitinases, cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into N-acetylglucosamine (NAG). NAGase from prawn (Penaeus vannamei) is involved in digestion and molting processes. The investigation of enzymatic properties, functional groups and catalytic mechanism is an essential mission to its commercial application. Bromacetic acid (BrAc) is a specific modifier for the histidine residue in specific condition. In this paper, the effect of BrAc on prawn NAGase activity for the hydrolysis of pNP-NAG has been investigated. The results showed that BrAc can reversibly and non-competitively inhibit the enzyme activity at appropriate concentrations and the value of IC(50) was estimated to be 17.05+/-0.65 mM. The inhibition kinetics of the enzyme by BrAc has been studied using the kinetic method of the substrate reaction. And the inhibition model was set up and the microscopic rate constants for the reaction of the inhibitor with free enzyme and the enzyme-substrate complexes were determined for inactivation and reactivation. The rate constant of the forward inactivation (k(+0)), which is 1.25 x 10(-3)s(-1), is about eight times as much as that of the reverse reactivation (k(-0)), which is 1.64 x 10(-4)s(-1). Therefore, when the BrAc concentration is sufficiently large, the enzyme is completely inactivated.