Irreversible inhibitory kinetics of mercuric ion on N-acetyl-β-d-glucosaminidase from Nile tilapia (Oreochromis niloticus)

•Hg2+ significantly decreases thermal and pH stability of tilapia NAGase.•The inhibition rate constants are calculated.•The substrate provides great protection to tilapia NAGase from Hg2+ inhibition.•The results are useful for evaluation of mercuric toxicity in tilapia reproduction.

N-acetyl-β-d-glucosaminidase (EC 3.2.1.52, NAGase), hydrolyzes dimers or trimers of N-acetyl-β-d-glucosamine (NAG) into monomers and is shown to be important for the reproduction of male animals. NAGase is purified from the spermary of Nile tilapia, and its enzyme activity can be strongly inhibited by mercuric chloride (HgCl2). In this paper, we determined the kinetics of HgCl2-mediated inhibition of NAGase, and our results showed that it was irreversible inhibition with an IC50 value at 2.70 ± 0.02 μM. Moreover, Hg2+ reduced the thermal and pH stability of the enzyme. We determined the inhibition kinetics of Hg2+ by using the kinetic method of substrate reaction. With this inhibition model, the microscopic rate constants for the reaction of Hg2+ with free enzyme (k1) and the enzyme–substrate complex (k′1k′1) were determined to be 4.42 × 10−4 mM−1 s−1 and 7.06 × 10−5 mM−1 s−1, respectively, indicating that the presence of substrate can protect NAGase from Hg2+ inhibition.