摘要
以锯缘青蟹(Scytta Serrata)内脏为材料,通过含0.2 mol/LNaCl的0.01 mol/LTris-HCl(pH 7.5)缓冲液抽提、硫酸铵分级沉淀、Sephadex G-100凝胶柱过滤层析、DE-32离子交换柱层析,分离提纯NAGase(NAGase),获得比活力为7,990U/mg的电泳单一纯NAGase制剂(NAGase,EC 3.2.1.52)。本文以此酶制剂为对象展开以下几方面的研究。
研究过氧化氢对锯缘青蟹NAGase活力的影响及抑制动力学。其结果表明,过氧化氢对该酶有显著的抑制作用,随着过氧化氢浓度的增高,酶活力呈指数下降,测出可使酶活力下降50%的过氧化氢浓度(抑制半衰期,IC_(50))为0.115 mol/L。低浓度过氧化氢对该酶的抑制过程显示为可逆效应。采用底物反应动力学方法研究过氧化氢对该酶的抑制作用动力学、建立动力学模型,测定游离酶(E)和酶-底物络合物(ES)的微观抑制速度常数k_(+0)和K′_(+0)并加以比较。实验结果(k_(+0)＞k′_(+0))表明底物对酶被过氧化氢的抑制作用有一定的保护作用。过氧化氢可能是通过氧化酶活性中心功能基团而导致酶活性的丧失。该研究对了解NAGase的功能基团性质及酶的催化作用机理提供重要的实验基础。
研究脲对酶的效应,结果表明,脲对锯缘青蟹N-乙酰-β-D-氨基葡萄糖苷酶(NAGase)有失活作用,随着脲浓度的增大,酶活力呈指数下降,测定导致酶活力下降50%的脲浓度(失活半衰期,IC_(50))为0.63 mol/L。酶在脲溶液中的失活过程显示为可逆失活。用底物反应动力学方法考察酶在脲溶液中的失活动力学,测定游离酶(E)和酶-底物络合物(ES)在脲溶液中失活的微观速度常数,并比较游离酶(E)和酶-底物络合物(ES)的正向反应的微观失活速度常数,表明底物存在对酶被脲的失活作用有一定的保护作用。正向的失活速度常数k_(+0)和k′_(+0)随着脲浓度的增大而增大,而逆向的速度常数k_(-0)随着脲浓度的增大而减小。表明随着脲浓度的增大,酶变性越来越快,而活力恢复越来越难。
研究十六种氨基酸对酶的效应,结果表明:非极性氨基酸中Gly、Ala、leu、Phe对酶活力几乎没有作用,Val、Ile对酶略有激活,Val浓度为20 mmol/L使酶活力提高7.5%,Ile浓度为40 mmol/L,使酶活力提高10.5%。Pro,Met对酶活力略有抑制作用,当Pro浓度为40 mmol/L时分别使酶活力下降15.2%,当Met浓度为40 mmol/L时分别使酶活力下降9.8%.极性氨基酸Asn、Cys、Ser、Thr对酶的活力没有影响。带负电荷的酸性氨基酸Asp和Glu对酶有抑制作用,当Asp浓度为40 mmol/L时,使酶活力下降38%;当Glu浓度为40 mmol/L时,可使酶活力下降25%。带正电荷的碱性氨基酸His略有抑制,Lys和Arg抑制作用较强,研究Lys和Arg对酶催化pNP-NAG水解反应的抑制机理,并测定其抑制常数,结果表明Lys和Arg的对酶的抑制均表现为可逆效应,抑制类型均为反竞争性抑制,其Kis分别为5.29 mmol/L和3.76 mmol/L。
研究对-氯汞苯甲酸(pCMB)对NAGase的化学修饰作用,巯基是酶活性的必须基团。氯化汞对锯缘青蟹NAGase抑制动力学研究,结果证明:当汞离子浓度小于1.0 lamol/L时,汞离子对剩余酶活力的作用表现为可逆反应,汞离子对酶的抑制机理属于竟争性抑制类型。通过汞离子对酶抑制作用的动力学模型的建立,测定游离酶(E)和酶.底物络合物(ES)在氯化汞溶液中失活的微观速度常数,研究汞离子浓度与表观速度常数之间的关系,结果证明:仅一分子氯化汞结合到酶分子活性中心就可导致酶活性的丧失。实验结果提示半胱氨酸残基位于酶活性中心,是酶分子的必须基团。
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(H_2O_2) can inhibit the enzyme activity obviously.The value of IC_(50),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 IC_(50)(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(HgCl_2) 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(Hg~(2+)) 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 Hg~(2+) 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 Hg~(2+) concentration has been studied and the result shows that only one molecule of HgCl_2 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.
引文
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