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首页> 外文期刊>Biochemistry >Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase.
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Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase.

机译：鉴定人线粒体支链氨基转移酶中CXXC基序的过氧化物敏感性氧化还原开关。

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摘要

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.
机译：人线粒体支链氨基转移酶同工酶（hBCATm）必须存储在还原性环境中才能保持活性。用巯基试剂氧化或标记hBCATm会导致酶抑制。在这项研究中，我们调查了hBCATm对这些试剂的敏感性的结构和生化基础。 hBCATm以其天然形式具有两个反应性半胱氨酸残基，使用碘化的β-（4-羟苯基）乙基马来酰亚胺将其鉴定为Cys315和Cys318。它们位于同型二聚体的大结构域中，距活性位点约10A。晶体结构显示出Cys315和Cys318之间存在巯基硫醇盐氢键的证据。在氧化条件下，由于硫原子之间的距离很短（3.09-3.46 A），这些半胱氨酸残基可以合理地形成二硫键，仅需减少1.1-1.5A。Cys315除了通过锚固Tyr173发挥结构性作用外，它以酮亚胺的形式增加了对活性位点的访问，我们的证据表明这些半胱氨酸残基在hBCATm中充当氧化还原开关。对5,5'-二硫代双（2-硝基苯甲酸）标记的hBCATm的电喷雾电离质谱分析和UV-Vis光谱研究表明，在标记过程中，蛋白质的显着部分形成了亚单位内二硫键。此外，已经确定hBCATm与H 2 O 2的反应消除了其活性，并导致在Cys315和Cys318之间形成亚单位内二硫键。加入二硫苏糖醇完全逆转了氧化并恢复了活性。因此，结果表明过氧化物敏感的CXXC中心对hBCATm活性具有氧化还原相关的调节作用。未来的研究将确定该中心是否在体内支链氨基酸代谢的调节中发挥作用。

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