Studies On Inhibitory Of Effectors On N-Acetyl-β-D-glucosaminidase From Scylla Serrata

N-Acetyl-β-D-glucosaminidase (NAGase, EC 3.2.1.52) was purified from viscera of green crab (Scylla serrata) and determined to be homogeneous by 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.(1) The effects of several metal ions on the enzyme activity had been studied. The results show that: Li⁺, Na⁺ and K⁺ have no effects on the activity of NAGase from Scylla serrata, while Ag+ can inhibit the enzyme activity with the inhibitor concentration leading to 50% of enzyme activity lost (IC₅₀) were estimated to be 2.32 mmol/L. Mg²⁺, Ca²⁺, Ba²⁺, Co²⁺ and Mn²⁺ can activate the enzyme. Mg²⁺, Ca²⁺ and Ba²⁺ can activate the enzyme with the order of Ca²⁺ > Ba²⁺ > Mg²⁺ in the intensity, while Mn²⁺ has a little active effects on the enzyme. The effect of Co²⁺ activate the enzyme at the concentrations lower than 4 mmol/L but inhibit it at higher concentrations. Cd²⁺, Pb²⁺ and Hg²⁺ inhibit the enzyme activity in different degree. The effects of Cd²⁺ and Pb²⁺ on the enzyme activity are reversible. The inhibition type of Cd²⁺ and Pb²⁺ on the enzyme belong to be mix-typed. The combination constants of Cd²⁺ with the free enzyme(KI)and the enzyme-substrate complex (KIS) were determined to be 23.9 and 5.0 mmol/L, while the values of KI and KIS of Pb²⁺ were determined to be 0.70 and 6.22 mmol/L. The effect of Hg²⁺ is irreversible. Conformational changes of the enzyme in different concentrations of Pb²⁺ and Hg²⁺ were measured by fluorescence spectra. The change of activity was more quickly than that of conformation on NAGase when inhibited by Pb²⁺and Hg²⁺. The inhibition kinetics of the enzyme by Pb²⁺ has been studied using the kinetic method of the substrate reaction during inhibition of enzyme activity previously described by Tsou. The microscopic rate constants for the reaction of this inhibitor with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of the enzyme against inhibition by Pb²⁺, Al³⁺ and Fe³⁺ also inhibit the enzyme activity. The effects of Al³⁺ and Fe³⁺ on the enzyme activity are reversible. The results show that the inhibition type of Fe³⁺on the enzyme belongs to be mix-typed. The values of KI and KIS of Fe³⁺ were determined to be 395.5 and 135.6μmol/L, respectively.(2) The effects of organic solvent on the enzyme activity had been studied. The...