Influence Of Effectors On N-Acetyl-β-D-glucosaminidase From Eriocheir Sinensis

N-Acetyl-β-D-glucosaminidase(NAGase,EC3．2．1．52) was purified from rivercrab (Eriocheir sinensis), by extraction with 0．01mol·L^-1 Tris-HCl buffer (pH7．5) containing 0．2 mol·L^-1 NaCl and 5 percent ethanol, then ammonium sulfate fraction,and then chromatography on DEAE-cellulose (DEAE-32) , Sephades G-100 andDEAE-32．The purified enzyme was a single band on polyacrylamide gelelectrophoresis with specific activity to be 4490．79U/mg. The subunit molecularweight were determined to be 121．21, 98．63 and 73．48 kD. The pI value wasdetermined to be 4．5 by isoelectric focusing.The optimum pH and optimumtemperature of the enzyme for the hydrolysis of p-Nitrophenyl-N-Acetyl-β-D-gluco-saminide (pNP-NAG) were investigated to be at pH5．5 and at 45℃, respectively.The result of the stability showed that the enzyme was stable at the pH range from 4．9to 9．3 and at the temperature below 45℃. The kinetic behavior of the enzyme in thehydrolysis of pNP-NAG followed Michaelis-Menten kinetics with K_m of0．357mmol·L and V_m of 10．409umol·L^-1·min^-1 at pH5．6 and 37℃, and theactivation energy was determined to be 76．50KJ/mol.The effects of different organic solwents on activity of the enzyme wereinvestigated. The results indicated that methanol and alcohol activated the enzymeat low concentrations, but they inhibited it at high concentration. Propanal,isopropanal, formaldehyde, acetone, phenol and SMOS inhibited the enzyme in varydegree. Methanol, alcohol and isopropanal were reversible competitive inhibitors.Theinhibition constants of free enzyme(K₁) of methanol, alcohol and isopropanal were0．288mol·L^-1 , 0．36mol·L^-1 and 0．48mmol·L^-1 , respectively. Formaldehyde, phenoland SMOS were reversible non-competitive inhibitors. The inhibition constants offree enzyme (K₁) of formaldehyde, phenol and SMOS were 0．288mol·L^-1 ,0．268mmol·L^-1 and 0．288mol·L^-1 , respectively. Propanal and acetone werereversible mixed-type inhibitors. The inhibition constants of free enzyme(K₁) andenzyme-substrate complex(K_IS) of propanal were determined to be 3．14mmol·L^-1 and 9．35mmol·L^-1,respectively. The inhibition constants of free enzyme (K₁) andenzyme-substrate complex(K_IS) of acetone were determined to be 0．195 mol·L^-1 and0．620 mol·L^-1 , respectively. Glutaraldehyde had no influence on the activity of theenzyme.The effects of metal ions on the enzyme were studied.The results showed thatNa⁺ activated the enzyme activity. Ba²⁺ and Ag⁺ activated the enzyme at low concentrations, but they inhibited it at high concentrations. Li⁺ and K⁺ ions inhibitedthe enzyme slightly. Mg²⁺, Cu²⁺, Pb²⁺, Zn²⁺, Mn²⁺ and Al³⁺ inhibited the enzyme.Mg²⁺, Cu²⁺ and Zn²⁺ were reversible non-competitive inhibitors. The inhibitionconstants of free enzyme (K₁) of Mg²⁺, Cu²⁺ and Zn²⁺ were 0．704 mol·L^-1 , 1．25mmol·L^-1 and 8．10mmol·L^-1 , respectively. Ag⁺ was reversible un-competitiveinhibitor. The inhibition constants(K₁) was 204．508mmol·L^-1 . Pb²⁺ was reversiblemixed-type inhibition.The inhibition constants of free enzyme(K₁) andenzyme-substrate complex(K_IS) of Pb²⁺ were determined to be 10．44mmol·L^-1 and 2．217mmol·L^-1 , respectively. Ca²⁺ had no influence on the activityof the enzyme.The effects of 12 kinds of amino acids on the enzyme were studied. L-Gly,L-Val, L-Thr, L-Ala, L-Phe and L-Ser had no influence on the activity of theenzyme in range of the certain concentrations. L-Gln, L-Met, L-Pro, L-Arg, L-Hisand L-Lys inhibited the enzyme in vary degree. L-Pro was reversible non-competitiveinhibitor. The inhibition constants of free enzyme (K₁) of L-Pro was 702．87mmol·L^-1 .L-Arg, L-His and L-Lys were reversible un-competitive inhibitors. The inhibitionconstants of free enzyme (K₁) of L-Arg, L-His and L-Lys were 1．682mmol·L^-1 ,85．81mmol·L^-1 and 1．517 mmol·L^-1 , respectively.The SO-3^2- which was brought because of the eutrophication could inhibit theenzyme activity obviously.lt was reversible un-competitive inhibitor. The inhibitionconstants of free enzyme(K₁) of SO₃^2- was 253．995mmol·L^-1 . The NO₂^-activatedthe enzyme activity when its concentration was in the range from 0 mmol·L^-1 to10mmol·L^-1 .The effect of hydrogen peroxide on the enzyme was studied.The inhibitionmechanism of hydrogen peroxide was reversible un-competitive mechanism.Theinhibition constants of free enzyme(K₁) of hydrogen peroxide was determined to be136．78mmol·L^-1 .Urea was reversible mixed-type inhibitor. The inhibition constants of freeenzyme(K₁) and enzyme-substrate complex(K_IS) of urea were determined to be379．02mmol·L^-1 and 1．182 mol·L^-1 , respectively.