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首页> 外文期刊>Biochemistry >Biochemical and Electrochemical Characterization of Two Variant Human Short-Chain Acyl-CoA Dehydrogenases
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Biochemical and Electrochemical Characterization of Two Variant Human Short-Chain Acyl-CoA Dehydrogenases

机译：两种变体人类短链酰基辅酶A脱氢酶的生化和电化学表征。

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Short-chain Acyl-CoA dehydrogenase(hSCAD)catalyzes the first matrix step in the mitochondrial beta-oxidation cycle with optimal activity toward butyryl-and hexanoyl-CoA.Two common variants of this enzyme encoding G185S and R147W substitutions have been identified at an increased frequency compared to the general population in patients with a wide variety of clinical problems,but functional studies of the purified mutant enzymes have shown only modestly changed kinetic properties.Moreover,both amino acid residues are located quite far from the catalytic pocket and the essential FAD cofactor.To clarify the potential relationship of these variants to clinical disease,we have further investigated their thermodynamic properties using spectroscopic and electrochemical techniques.Purified R147W hSCAD exhibited almost identical physical and redox properties to wild-type but only half of the specific activity and substrate activation shifts observed in wild-type enzyme.In contrast,the G185S mutant proved to have impairments of both its kinetic and electron transfer properties.Spectroelectro-chemical studies reveal that G185S binding to the substrate/product couple produces an enzyme potential shift of only +88 mV,which is not enough to make the reaction thermodynamically favorable.For wild-type hSCAD,this barrier is overcome by a negative shift in the substrate/product couple midpoint potential,but in G185S this activation was not observed.When G185S was substrate bound,the midpoint potential of the enzyme actually shifted more negative.These results provide valuable insight into the mechanistic basis for dysfunction of the common variant hSCADs and demonstrate that mutations,regardless of their position in the protein structure,can have a large impact on the redox properties of the enzyme.
机译：短链酰基辅酶A脱氢酶（hSCAD）催化线粒体β-氧化循环的第一步，对丁酰基和己酰基辅酶A具有最佳活性，已发现该酶编码G185S和R147W取代的两个常见变体具有广泛临床问题的患者与普通人群相比频率较高，但是对纯化的突变酶的功能研究仅显示了适度变化的动力学特性。此外，两个氨基酸残基都位于远离催化口袋和必需FAD的位置为了阐明这些变异体与临床疾病的潜在关系，我们使用光谱学和电化学技术进一步研究了它们的热力学性质。纯化的R147W hSCAD表现出与野生型几乎相同的物理和氧化还原特性，但只有一半的比活和底物在野生型酶中观察到活化转移。相反，G185S突变光谱化学研究表明，G185S与底物/产物对的结合产生的酶电位偏移仅为+88 mV，不足以使反应在热力学上有利。对于野生型hSCAD，该障碍可通过底物/产物偶数中点电位的负移来克服，但在G185S中未观察到这种激活。当G185S被底物结合时，酶的中点电位实际上移得更负。结果为常见变异hSCAD功能障碍的机理基础提供了有价值的见解，并证明了突变，无论其在蛋白质结构中的位置如何，都可能对酶的氧化还原特性产生重大影响。

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