The stability of G6PD is affected by mutations with different clinical phenotypes.
The stability of G6PD is affected by mutations with different clinical phenotypes.
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DOI：
10.3390/ijms151121179
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发表时间：
2014-11-17
影响因子：
5.6
通讯作者：
Enríquez-Flores S
中科院分区：
文献类型：
--
作者：
Gómez-Manzo S;Terrón-Hernández J;De la Mora-De la Mora I;González-Valdez A;Marcial-Quino J;García-Torres I;Vanoye-Carlo A;López-Velázquez G;Hernández-Alcántara G;Oria-Hernández J;Reyes-Vivas H;Enríquez-Flores S
葡萄糖-6-磷酸脱氢酶（G6 PD）缺乏症是世界范围内最常见的酶缺乏症，可导致多种疾病，严重程度从最轻微（IV类）到最严重（I类）不等。为了将G6 PD中的突变位点与所产生的表型相关联，我们研究了四种天然存在的G6 PD变体：尤卡坦、纳什维尔、巴利亚多利德和墨西哥城。为此，我们开发了一种成功的过表达方法，该方法构成了用于获得和表征这些酶的更容易和更精确的方法。所有研究变体的kcat（催化常数）均低于野生型。结构刚性可能是原因，突变的最明显后果是它们对蛋白质稳定性和折叠的影响，如可以从蛋白质产率、T50（保留其原始活性的50%的温度）值和疏水区域的差异中观察到的。对应于更严重表型的突变与结构NADP+区域相关。这对于III类和II类变体清楚地观察到，其随着NADP+的增加而变得更加热稳定，而I类变体保持热不稳定。突变产生排斥电荷，在尤卡坦变异体的情况下，促进C-末端的增加的紊乱，从而影响NADP+的结合，导致酶不稳定。
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The kcat (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T50 (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP+ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP+, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP+, leading to enzyme instability.
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作者：
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影响因子：
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