Catalytic mechanism of S-adenosylhomocysteine hydrolase:: Roles of His 54, Asp130, Glu155, Lys185, and Asp189
Catalytic mechanism of S-adenosylhomocysteine hydrolase:: Roles of His 54, Asp130, Glu155, Lys185, and Asp189
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DOI：
10.1016/j.biocel.2005.06.009
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发表时间：
2005-11-01
影响因子：
4
通讯作者：
Takusagawa, F
中科院分区：
文献类型：
--
作者：
Yamada, T;Takata, Y;Takusagawa, F
S-腺苷高半胱氨酸水解酶（AdoHcyase）催化S-腺苷高半胱氨酸（AdoHCy）水解形成腺苷和高半胱氨酸。具有弱催化活性（0.1%）的K185 N突变酶的晶体结构已经在2.8埃分辨率下确定，并且支持先前预测的机制[Takata，Y.，Yamada，T.，黄，Y.，Komoto，J.，Gomi，T.，小川，H.，Fujioka，M.，& Takusagawa，F.（2002年）的报告。S-腺苷高半胱氨酸水解酶的催化机制。Asp-130、Lys-185、Asp-189和Asn-190的定点诱变。J. Biol. Chem. 277，22670-22676]。突变的酶具有开放和闭合构象之间的中间结构，分别在无底物的酶和抑制剂复合物中观察到。将H54、H300和H352分别突变为天冬酰胺，以鉴定组氨酸残基在催化中的作用。H54 N、H300 N和H354 N突变酶的动力学数据表明，H54是充当一般酸/碱以切割H2 Hcy的C5 '-S-D键的氨基酸残基。E155 Q突变的酶保留了大部分催化活性（31%），而E155 D突变的酶失去了大部分催化活性（0.3%）。突变酶的NADH积累测定表明，C3 ′-氧化和C4 ′-质子夺取是一个协同事件，而C5 ′-S-D键断裂是一个独立事件。C4 ′-质子交换测定表明，当α-Hcy在活性中心转化为3 ′-酮基-4 ′，5 ′-脱氢-Ado时，酶具有开放构象。结合前人的研究结果，对该酶的催化机理进行了详细的阐述。K185促进C3 '-氧化，D130提取C4'-质子，D189和E155充当协调的C3 '-氧化和C4'-质子提取之间的通信者，并且H54充当通用酸以切割C5 '-SD键。(c)2005年由Elsevier Ltd.出版
S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine (AdoHcy) to form adenosine and homocysteine. The crystal structure of the K185N mutated enzyme, which has weak catalytic activity (0.1%), has been determined at 2.8 angstrom resolution and supports the previously predicted mechanism [Takata, Y., Yamada, T., Huang, Y., Komoto, J., Gomi, T., Ogawa, H., Fujioka, M., & Takusagawa, F. (2002). Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J. Biol. Chem. 277, 22670-22676]. The mutated enzyme has an intermediate structure between the open and closed conformation, observed in the substrate-free enzyme and in the inhibitor complexes, respectively. H54, H300, and H352 were mutated to asparagine, respectively, to identify the roles of the histidine residues in catalysis. The kinetic data of H54N, H300N, and H354N mutated enzymes suggest that H54 is the amino acid residue that acts as a general acid/base to cleave the C5'-S-D bond of AdoHcy. The E155Q mutated enzyme retained a large portion of the catalytic activity (31%), while the E155D mutated enzyme lost most of it (0.3%). The NADH accumulation measurements of the mutated enzymes indicated that the C3'-oxidation and the C4'-proton abstraction are a concerted event and the C5'-S-D bond cleavage is an independent event. The C4'-proton exchange measurements indicate that the enzyme has an open conformation when AdoHcy is converted to 3'-keto-4', 5'-dehydro-Ado in the active site. With the results of this study and those of the previous studies, a detailed catalytic mechanism of AdoHcyase is described. K185 facilitates the C3'-oxidation, D130 abstracts the C4'-proton, D189, and E155 act as a communicator between the concerted C3'-oxidation and C4'-proton abstraction, and H54 plays as a general acid to cleave the C5'-SD bond of AdoHcy. (c) 2005 Published by Elsevier Ltd.