Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease.
Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease.
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人天冬氨酸酰化酶的表征：导致卡纳万病的脑酶。
DOI：
10.1021/bi052608w
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Viola,RonaldE
中科院分区：
文献类型：
--
作者：
LeCoq,Johanne;An,Hyun-Joo;Lebrilla,Carlito;Viola,RonaldE
天冬氨酸氨基转移酶催化N-乙酰天冬氨酸(NAA)脱乙酰基生成乙酸酯和l-天冬氨酸，是唯一一种能有效代谢NAA的脑酶。虽然这种酶反应的确切作用尚未完全阐明，但NAA的代谢似乎是形成髓鞘脂类所必需的，该酶的缺陷会导致Canavan病，一种致命的神经疾病。大肠杆菌表达的天冬氨酸酰基酶的低催化活性和固有的不稳定性表明，需要一个合适的真核表达系统来生产功能齐全的成熟酶。人天冬氨酸氨基转移酶已在巴斯德毕赤酵母中成功表达。而表达产物的产量比INE低。Coli中，纯化后的酶明显更稳定。这种酶形式具有相同的底物专一性，但活性是THE的150倍。Coli表达的酶。用质谱仪测定纯化的酶的相对分子质量比预测的要高，这表明存在一些翻译后修饰。天冬氨酸酯酶的脱糖基化或糖基化位点的突变会导致酶的稳定性降低和催化活性降低。去除了一种碳水化合物成分，并用质谱学对其进行了表征。除了这种碳水化合物部分，这种酶还被证明每个亚基含有一个锌原子。去除锌的络合研究导致可逆的催化活性丧失，从而确立了天冬氨酰基酶作为锌金属酶的地位。
Aspartoacylase catalyzes the deacetylation ofN-acetylaspartic acid (NAA) to produce acetate andl-aspartate and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids, and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with theEscherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed inPichia pastoris. While the expression yields are lower than inE. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity but is 150-fold more active than theE. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some post-translational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc result in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme.