Specific labeling of the essential cysteine residue of L-methionine gamma-lyase with a cofactor analogue, N-(bromoacetyl)pyridoxamine phosphate.
Specific labeling of the essential cysteine residue of L-methionine gamma-lyase with a cofactor analogue, N-(bromoacetyl)pyridoxamine phosphate.
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使用辅因子类似物 N-(溴乙酰基)吡哆胺磷酸盐特异性标记 L-蛋氨酸 γ-裂合酶的必需半胱氨酸残基。
DOI：
10.1021/bi00405a029
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
K. Soda
中科院分区：
文献类型：
--
作者：
T. Nakayama;N. Esaki;H. Tanaka;K. Soda
来自恶臭假单胞菌的L-甲硫氨酸γ-裂解酶由四条相同的多肽链组成，每个亚基含有四个半胱氨酰残基。我们发现其中一个是通过它与2-硝基-5-硫氰基苯甲酸的特定氰化反应而催化必需的。我们已经用N-（溴乙酰基）吡哆胺5 '-磷酸（BAPMP）证明了它的重要性，BAPMP是一种辅因子类似物，也是一种亲和标记剂。动力学数据表明，脱辅基酶形成一个二元复合物与BAPMP之前，共价结合。失活的化学计量为1 mol BAPMP/亚基。我们已经证明，用BAPMP修饰的半胱氨酸残基与用[14 C]碘乙酸特异性标记的半胱氨酸残基相同。通过自动Edman降解测定含有必需半胱氨酸残基和5 '-磷酸吡哆醛结合的赖氨酸残基的肽的氨基酸序列。
L-Methionine gamma-lyase from Pseudomonas putida is composed of four identical polypeptide chains and contains four cysteinyl residues per subunit. We have found one of them catalytically essential by its specific cyanylation with 2-nitro-5-thiocyanobenzoic acid. We have shown its essentiality also with N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP), which is a cofactor analogue and also an affinity-labeling agent. The kinetic data show that the apoenzyme forms a binary complex with BAPMP prior to covalent binding. The stoichiometry of inactivation was 1 mol of BAPMP per subunit. We have shown that the cysteine residue modified with BAPMP is identical with that labeled specifically with [14C]iodoacetic acid. The amino acid sequences of the peptides containing the essential cysteine residue and the lysine residue to which pyridoxal 5'-phosphate is bound were determined by automated Edman degradation.