Bross P, Andresen B S, Winter V, Kräutle F, Jensen T G, Nandy A, Kølvraa S, Ghisla S, Bolund L, Gregersen N
Dept. of Clinical Chemistry, Arhus Kommunehospital and Skejby Sygehus, Arhus, Denmark.
Biochim Biophys Acta. 1993 Oct 20;1182(3):264-74. doi: 10.1016/0925-4439(93)90068-c.
The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism underlying MCAD deficiency caused by the prevalent K304E mutation. Depending on which of the three amino acids--lysine (wild-type), glutamic acid (K304E) or glutamine (K304Q) are present at position 304 of the mature polypeptide, three different patterns were observed in our assay system: (i) solubility, tetramer formation and yield of enzyme activity of wild-type MCAD is largely independent of GroESL co-overexpression; (ii) the larger part of the K304Q mutant is insoluble without and solubility is enhanced with GroESL co-overexpression; solubility correlates with the amount of tetramer detected and the enzyme activity measured as observed for the wild-type protein. (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected in Western blotting. In a first attempt to estimate the specific activity, we show that tetrameric K304E and K304Q mutant MCAD display a specific activity in the range of the wild-type enzyme. Taken together, our results strongly suggest, that the K304E mutation primarily impairs the rate of folding and subunit assembly. Based on the data presented, we propose that lysine-304 is important for the folding pathway and that an exchange of this amino acid both to glutamine or glutamic acid leads to an increased tendency to misfold/aggregate. Furthermore, exchange of lysine-304 with an amino acid with negative charge at position 304 (glutamic acid) but not with a neutral charge (glutamine) negatively affects conversion to active tetramers. A possible explanation for this latter effect--charge repulsion upon subunit docking--is discussed.
为了研究由常见的K304E突变导致的中链酰基辅酶A脱氢酶（MCAD）缺乏症的分子机制，分析了细菌伴侣蛋白GroEL和GroES共过表达对三种异源表达的人MCAD变体的溶解度、四聚体形成和酶活性的影响。根据成熟多肽第304位存在的三种氨基酸中的哪一种——赖氨酸（野生型）、谷氨酸（K304E）或谷氨酰胺（K304Q），在我们的检测系统中观察到三种不同的模式：（i）野生型MCAD的溶解度、四聚体形成和酶活性产量在很大程度上与GroESL共过表达无关；（ii）K304Q突变体的大部分在没有GroESL共过表达时是不溶的，而溶解度随着GroESL共过表达而增强；溶解度与检测到的四聚体数量和测量的酶活性相关，这与野生型蛋白的情况相同。（iii）K304E突变体的溶解度与K304Q突变体对GroESL的反应方式相似，但观察到的四聚体数量和测量的酶活性与在蛋白质印迹中检测到的可溶性K304E MCAD蛋白数量不相关。在首次尝试估计比活性时，我们表明四聚体K304E和K304Q突变体MCAD的比活性在野生型酶的范围内。综上所述，我们的结果强烈表明，K304E突变主要损害折叠速率和亚基组装。基于所呈现的数据，我们提出赖氨酸-304对折叠途径很重要，并且该氨基酸与谷氨酰胺或谷氨酸的交换都会导致错误折叠/聚集的倾向增加。此外，在第304位用带负电荷的氨基酸（谷氨酸）而非中性电荷的氨基酸（谷氨酰胺）替换赖氨酸-304会对转化为活性四聚体产生负面影响。讨论了后一种效应——亚基对接时的电荷排斥——的可能解释。
