Identification of methionine-110 as the residue covalently modified in the electrophilic inactivation of D-amino-acid oxidase by O-(2,4-dinitrophenyl) hydroxylamine.
Identification of methionine-110 as the residue covalently modified in the electrophilic inactivation of D-amino-acid oxidase by O-(2,4-dinitrophenyl) hydroxylamine.
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鉴定出蛋氨酸-110 是 D-氨基酸氧化酶被 O-(2,4-二硝基苯基)羟胺亲电失活过程中共价修饰的残基。
DOI：
10.1021/bi00380a034
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Massey,V
中科院分区：
文献类型：
--
作者：
D'Silva,C;WilliamsJr,CH;Massey,V
修订稿于 1986 年 11 月 7 日收到 摘要：< 9-(2, 4-二硝基苯基) 羟胺与 D-氨基酸氧化酶的反应导致完全失活，可通过竞争性抑制剂苯甲酸盐进行保护 [D’Silva, C., Williams, C. H., Jr., & Massey, V.(1986) Biochemistry 25, 5602-5608]，修饰后的残基已被鉴定为蛋氨酸-110。在苯甲酸盐不存在和存在的情况下修饰的 D-氨基酸氧化酶的胰蛋白酶消化物的差示高效液相色谱图谱允许分离对应于残基 100-115 并称为 T6-T7 的单个含甲硫氨酸的胰蛋白酶肽。在未修饰的酶中，涉及 Arg-108 的键很容易断裂，并且 T6 和 T7 被分离。用羧肽酶 Y 短暂处理肽 T6-T7，释放残基 112-115，并且以良好的收率分离出残余肽。用羧肽酶 Y 进一步处理该肽（残基 100-111），释放出 Val 和未知氨基酸，并与合成制备的 5-氨基甲硫氨酸锍盐共迁移。用二硫苏糖醇处理后，未知化合物和 5-氨基甲硫氨酸分解为甲硫氨酸。
Revised Manuscript Received November 7, 1986 abstract: The reaction of< 9-(2, 4-dinitrophenyl) hydroxylamine with D-amino-acid oxidaseleads to complete inactivation which can be protected against by the competitive inhibitor benzoate [D’Silva, C., Williams, C. H., Jr., & Massey, V.(1986) Biochemistry 25, 5602-5608], The residue modified has been identified as methionine-110. Differential high-performance liquid chromatography mapping oftryptic digests of D-amino-acid oxidase modified in the absence and presence of benzoate allows the isolation of a single methionine-containing tryptic peptide corresponding to residues 100-115 and referred to as T6-T7. In unmodified enzyme, the bond involving Arg-108 is readily cleaved and T6 and T7 are isolated. Brief treatment of peptide T6-T7 with carboxypeptidase Y released residues 112-115, and the residual peptide was isolated in good yield. Further treatment of this peptide (residues 100-111) with carboxypeptidase Y released Val and an unknown amino acid that comigrated with synthetically prepared 5-aminomethionine sulfonium salt. The unknown compound and 5-aminomethionine break down to methionine on treatment with di-thiothreitol.