Inactivation of monomeric sarcosine oxidase by reaction with N-(cyclopropyl)glycine.
Inactivation of monomeric sarcosine oxidase by reaction with N-(cyclopropyl)glycine.
复制标题
通过与 N-(环丙基)甘氨酸反应灭活单体肌氨酸氧化酶。
DOI：
10.1021/bi001421w
复制
发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Jorns,MS
中科院分区：
文献类型：
--
作者：
Zhao,G;Qu,J;Davis,FA;Jorns,MS
单体肌氨酸氧化酶（MSOX）催化肌氨酸（N-甲基甘氨酸）的氧化去甲基化，并含有共价结合的黄素腺嘌呤二核苷酸（FAD）。本研究表明，N-（环丙基）甘氨酸（CPG）是一种基于机制的抑制剂。CPG与MSOX形成电荷转移复合物，在有氧条件下反应产生共价修饰的还原黄素（λmax= 422 nm，ε422= 3.9 mM-1cm-1），伴随着酶活性的损失。CPG修饰的黄素以慢8倍的速率转化为1，5-二氢-FAD（EFADH 2），其与氧气快速反应以再生未修饰的氧化酶。因此，CPG修饰的MSOX在有氧条件下达到CPG依赖性稳态浓度，并在去除过量试剂后恢复为未修饰的酶。在厌氧条件下未观察到活性损失，其中EFADH 2在低CPG浓度下完成的反应中形成。CPG修饰的酶的有氧变性以类似于产生1，5-二氢-FAD的厌氧变性反应的速率产生未修饰的氧化黄素。CPG修饰的黄素可以用硼氢化物还原，这是一种在去除过量的CPG或酶变性后阻止转化为未修饰的黄素的反应。讨论了CPG改性黄素的结构和可能的化学失活机理。
Monomeric sarcosine oxidase (MSOX) catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound flavin adenine dinucleotide (FAD). The present study demonstrates thatN-(cyclopropyl)glycine (CPG) is a mechanism-based inhibitor. CPG forms a charge transfer complex with MSOX that reacts under aerobic conditions to yield a covalently modified, reduced flavin (λmax= 422 nm, ε422= 3.9 mM-1cm-1), accompanied by a loss of enzyme activity. The CPG-modified flavin is converted at an 8-fold slower rate to 1,5-dihydro-FAD (EFADH2), which reacts rapidly with oxygen to regenerate unmodified, oxidized enzyme. As a result, CPG-modified MSOX reaches a CPG-dependent steady-state concentration under aerobic conditions and reverts back to unmodified enzyme upon removal of excess reagent. No loss of activity is observed under anaerobic conditions where EFADH2is formed in a reaction that goes to completion at low CPG concentrations. Aerobic denaturation of CPG-modified enzyme yields unmodified, oxidized flavin at a rate similar to the anaerobic denaturation reaction, which yields 1,5-dihydro-FAD. The CPG-modified flavin can be reduced with borohydride, a reaction that blocks conversion to unmodified flavin upon removal of excess CPG or enzyme denaturation. The possible chemical mechanism of inactivation and structure of the CPG-modified flavin are discussed.