Electrophilic amination of a single methionine residue located at the active site of D-amino acid oxidase by O-(2,4-dinitrophenyl)hydroxylamine.
Electrophilic amination of a single methionine residue located at the active site of D-amino acid oxidase by O-(2,4-dinitrophenyl)hydroxylamine.
复制标题
DOI：
10.1021/bi00367a039
复制
发表时间：
1986-09
期刊：
影响因子：
2.9
通讯作者：
C. D’Silva;C. Williams;V. Massey
中科院分区：
文献类型：
--
作者：
C. D’Silva;C. Williams;V. Massey
O-(2,4-二硝基苯基)羟胺是一种 D-氨基酸氧化酶的快速活性位点定向抑制剂：修饰可将胺基特异性掺入可接近的亲核残基中，同时释放 2,4-二硝基苯酚。该反应被竞争性抑制剂苯甲酸酯阻止，表明是活性位点定向反应。在pH 7.0 下观察到每个酶结合的黄素腺嘌呤二核苷酸单体的化学计量为1-1.5 mol 胺残基。氨基酸和序列分析表明His-217不是修饰反应的目标。修饰对 pH 的依赖性、对氨基酸上存在的官能团的模型研究以及对胺化酶的硫解研究共同表明修饰位于酶活性位点处或附近的甲硫氨酸残基上。胺化酶虽然在光谱上与天然酶相似，但在 455 nm 黄素吸收中表现出 7-9 nm 蓝移。苯甲酸盐会干扰胺化酶的谱，但相对于天然酶的结合要弱得多（pH 8.0 时 Kd 大约高 300 倍）。
O-(2,4-Dinitrophenyl)hydroxylamine is a rapid active-site-directed inhibitor of D-amino acid oxidase: modification results in specific incorporation of an amine group into an accessible nucleophilic residue with concomitant release of 2,4-dinitrophenol. The reaction is prevented by the competitive inhibitor benzoate, indicating an active-site-directed reaction. A stoichiometry of 1-1.5 mol of amine residues per enzyme bound flavin adenine dinucleotide monomer was observed at pH 7.0. Amino acid and sequence analyses show that His-217 is not the target of the modification reaction. Dependence of the modification on pH, model studies on functional groups present on amino acids, and thiolysis studies on aminated enzyme collectively indicate that the modification is located on a methionine residue at or near the active site of the enzyme. Aminated enzyme, although spectrally similar to native enzyme, exhibits a 7-9-nm blue shift in the 455-nm flavin absorption. Benzoate perturbs the spectrum of aminated enzyme, but binding relative to native enzyme is much weaker (Kd ca. 300 times greater at pH 8.0).