N-乙酰鸟氨酸脱酰基酶的化学修饰
首发时间:2007-10-02
摘要:采用PMSF、NBS、DTNB、DEPC、WRK五种化学试剂,选择性修饰N-乙酰鸟氨酸脱酰基酶中丝氨酸的羟基、色氨酸的吲哚基、半胱氨酸的巯基、组氨酸的咪唑基、天冬氨酸和谷氨酸的羧基,研究氨基酸侧链基团与酶活性中心的关系。结果显示,以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可能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形成。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。
关键词: N-乙酰鸟氨酸脱酰基酶 活性中心 化学修饰 底物保护
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Effects of Protein Modification Reagents
Abstract:Acetylornithine deacetylase (NAOase) from E.coli was selectively modified by five chemical reagents such as phenylmethylsulfonylfluoride(PMSF),N-bromosuccinimide(NBS) ,5, 5 -dithio-bis-2-ni-trobenzonic-acid,(DTNB),diethypyro-carbonate(DEPC),N-ethyl-5-phenylisoxa zolium 3-sulfonate(WRK) respectively.It can be found that the enzyme activity was significantly decreased after modification of DEPC,WRK but oppositely the PMSF,NBS,DTNB did not obviously effect the enzyme activity. Study of the chemical modification demonstrated that the histidine residues, carboxyl groups are essential functional groups inside of the NAOase active site,at the same time suggested the tryptophan residues,Serine residues and cysteine residues are not located in the enzyme active site ,and didn’t directly effect the enzyme activity.Substrate N-acetyl-D,L- methionine may protect the enzyme, and the protection would be increased when its concentration increased.
Keywords: acetylornithine deacetylase, active center, chemical modification, substrate protection
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