Beyond the proton abstracting role of Glu-376 in medium-chain acyl-CoA dehydrogenase: influence of Glu-376-->Gln substitution on ligand binding and catalysis.
Beyond the proton abstracting role of Glu-376 in medium-chain acyl-CoA dehydrogenase: influence of Glu-376-->Gln substitution on ligand binding and catalysis.
复制标题
除了 Glu-376 在中链酰基辅酶 A 脱氢酶中的质子提取作用之外：Glu-376-->Gln 取代对配体结合和催化的影响。
DOI：
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发表时间：
2002
期刊：
影响因子：
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通讯作者：
Srivastava Dk
中科院分区：
文献类型：
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作者：
Gopalan Kv;Srivastava Dk
已知中链酰基辅酶A脱氢酶（MCAD）的活性位点残基Glu-376在还原半反应过程中从酰基辅酶A底物中提取α-质子。Glu-376→Gln（E376 Q）的位点特异性突变使酶的辛酰辅酶A依赖性还原半反应减慢约5个数量级，这是由于质子转移步骤的受损。为了测试Glu-376的羧基是否在酶催化过程中专门作为活性位点碱基（用于提取α-质子），我们对酶-配体相互作用和酶催化进行了详细的动力学研究，利用辛酰辅酶A/辛烯酰辅酶A作为生理底物/产物对，野生型和E376 Q突变酶作为催化剂。瞬时动力学数据表明，E376 Q突变不仅损害了酶结合FAD的辛酰辅酶A依赖性还原速率，而且损害了反应物结合FAD的缔合和解离速率。
The active site residue, Glu-376, of medium-chain acyl-CoA dehydrogenase (MCAD) has been known to abstract the α-proton from acyl-CoA substrates during the course of the reductive half-reaction. The site-specific mutation of Glu-376→Gln(E376Q) slows down the octanoyl-CoA-dependent reductive half-reaction of the enzyme by about 5 orders of magnitude due to impairment in the proton-transfer step. To test whether the carboxyl group of Glu-376 exclusively serves as the active site base (for abstracting the α-proton) during the enzyme catalysis, we undertook a detailed kinetic investigation of the enzyme−ligand interaction and enzyme catalysis, utilizing octanoyl-CoA/octenoyl-CoA as a physiological substrate/product pair and the wild-type and E376Q mutant enzymes as the catalysts. The transient kinetic data revealed that the E376Q mutation not only impaired the rate of octanoyl-CoA-dependent reduction of the enzyme-bound FAD, but also impaired the association and dissociation rates for the binding of the react...