The Inhibitory Kinetics Of N-Acetyl-β-D-glucosaminidase From Green Crab (Scylla Serrata)

N-Acetyl-β-D-glucosaminidase(NAGase,EC 3.2.1.52) was purified from viscera of green crab(Scylla serrata),by extraction with 0.01 mol/L Tris-HCl buffer(pH 7.5) containing 0.2 mol/L NaCl and ammonium sulfate fractionation,then chromatography on Sephadex G-100 and DEAE-cellulose(DE-32).The purified enzyme was a single band on polyacrylamide gel electrophoresis(PAGE) and SDS-PAGE with specific activity to be 7,990 U/mg.This purfied enzyme will be used in the following studies.The effect of hydrogen peroxide on N-acetyl-β-D-glucosarninidase(NAGase) from green crab was investigated.The results showed that hydrogen peroxide(H₂O₂) can inhibit the enzyme activity obviously.The value of IC₅₀,the inhibitor's concentration leading to 50%activity lost,was estimated to be 0.115 mol/L.The inhibition of the enzyme by hydrogen peroxide is a reversible reaction with remaining enzyme activity.The inhibitory kinetics of the enzyme by hydrogen peroxide was studied using the Tsou's method of the substrate reaction,and the microscopic rate constants of inhibition for the free enzyme and the enzyme-substrate complex were determined.Comparison of these rate constants was made,the result showed that k₊₀ is larger than k'₊₀,indicating a marked protective effect of the substrate on the inhibition reaction of this enzyme with hydrogen peroxide.The effect of urea on the enzyme was investigated.The results showed that urea can inactivate the enzyme activity,and the IC₅₀(inactivator's concentration leading to 50%activity lost) was estimated to be 0.63 mol/L.The inactivation belongs to be reversible reaction with remaining enzyme activity.The kinetics of inactivation of the enzyme in urea solutions has been studied using the Tsou's method of the substrate reaction,and the microscopic rate constants of inactivation for the free enzyme and the enzyme-substrate complex were determined.Comparison of these rate constants showed that k₊₀ is larger than k'₊₀,indicating a marked protective effect of the substrate on the inactivation reaction.Moreover,the value of forward inactivation rate constant of the enzyme(k₊₀ and k'₊₀) increased with increasing urea concentration while the value of reverse reaction(k_-0) decreased.The results suggested the inactivation of enzyme in higher concentration of urea would be more difficultly reversible.NAGase,catalyzes the cleavage of N-acetylglucosamine polymers,is widely distributed in animal tissues and in microorganisms.The effects of some amino acids on the enzyme activity were studied.The results showed that Gly,Ala,Asn,Cys,Ser, Thr,Leu and Phe had no effects;Val and Ile activated lightly;Pro,Met had inhibitory effects lightly,while Asp,Glu,Lys,Arg and His had inhibitory effects on the enzyme activity.The inhibitory effects of Lys and Arg were reversible with remaining enzyme activity and the inhibitory mechanisms were tested to be un-competitive types and their K_IS were determined to be 5.29 and 3.76 mmol/L,respectively.Chemistry modification of p-chloromercuribenzoate(PCMB) on the enzyme has been studied.The results show that sulfhydryl group is essential for the activity of the enzyme.Inhibitory kinetics of the enzyme by mercuric chloride(HgCl₂) has been studied using the kinetic method of the substrate reaction during inhibitor of enzyme. The kinetic results show that the inhibition of the enzyme by mercuric ion(Hg²⁺) at lower than 1.0μmol/L is a reversible reaction with residual activity and the inhibition belongs to be competitive.The inhibition kinetics model of Hg²⁺ on the enzyme was set up and the microscopic rate constants were determined and the data obtained were well fitted with the model.The relationship between the apparent rate constants and Hg²⁺ concentration has been studied and the result shows that only one molecule of HgCl₂ binds to the enzyme molecule to lead the enzyme lose its activity.The above results suggest that the cysteine residue is essential for activity and is situated at the active site of the enzyme.