N-乙酰鸟氨酸脱酰基酶活性位点的构成研究

Composition of Acetylomithine Deacetylase Active Site

目的:用生物信息学软件预测出N-乙酰鸟氨酸脱酰基酶的活性中心的金属离子结合位点。方法:选用DEPC、WRK、PMSF、NBS、DTNB 5种化学试剂选择性修饰N-乙酰鸟氨酸脱酰基酶中组氨酸、天冬氨酸、谷氨酸、丝氨酸、色氨酸和半胱氨酸;同时考察Co^(2+)、Fe^(3+)、Mg^(2+)、Mn^(2+)、Zn^(2+)、Ni^(2+)、Cu^(2+)等金属离子对酶活性的影响。结果:在DEPC、WRK修饰后,酶的活力明显下降,而PMSF、NBS、DTNB对酶的活力影响不大;说明组氨酸和酸性氨基酸为酶活性中心的必需氨基酸,而丝氨酸残基、色氨酸残基、半胱氨酸残基不参与酶活性中心的组成;Co^(2+)对酶反应有促进作用,验证了生物信息学的预测结果;底物N-乙酰-D,L-蛋氨酸对酶有较好的保护作用,保护作用随浓度增加而增加。结论:本研究为深入研究酶结构与功能的关系提供实验依据,为N-乙酰鸟氨酸脱酰基酶的工业应用提供理论参考。

Objective： To investigate the binding site of metal ion in active center of acetylornithice deacetylase （NAOase） by bioinformatics software. Methods： Histidine of acetylornithice deacetylase （NAOase） from E.coli was selectively modified by five chemical reagents： phenylmethyl-sulfonyl-fluoride （PMSF）, N-bromosuccinimide （NBS）, 5,5-dithio-bis-2-ni-trobenzonic- acid （DTNB）, diethypyro-carbonate （DEPC/ and N-ethyl-5-phenylisoxa zolium 3-sulfonate （WRK） respectively. Results： It is found that the enzyme activity was significantly decreased after modification with DEPC and WRK respectively, while the PMSF, NBS, DTNB did not have obvious effects on the enzyme activity. They demonstrated that the histidine residues and carboxyl groups are essential functional groups inside of the NAOase active site, but the tryptophan residues, serine residues and cysteine residues are not located in the enzyme active site and have no direct effects on enzyme activity. Substrate N-acetyl-D, L- methionine may protect the enzyme, and the protection would increase when its concentration increased. Conclusion： The research provid experiment foundation to lucubrate, the relation between enzyme structure and function, and offer theoretical reference to make use ofacetylornithice deacetylase in industry.