Medium-long-chain chimeric human acyl-CoA dehydrogenase: Medium-chain enzyme with the active center base arrangement of long-chain acyl-CoA dehydrogenase
Medium-long-chain chimeric human acyl-CoA dehydrogenase: Medium-chain enzyme with the active center base arrangement of long-chain acyl-CoA dehydrogenase
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DOI：
10.1021/bi960785e
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发表时间：
1996-09-24
期刊：
影响因子：
2.9
通讯作者：
Ghisla, S
中科院分区：
文献类型：
--
作者：
Nandy, A;Kieweg, V;Ghisla, S
通过将底物α-氢提取为H+来启动催化的催化必需的谷氨酸残基位于中链酰基-CoA脱氢酶（MCADH）中环JK上的位置376（成熟MCADH编号）。在长链酰基辅酶A脱氢酶（LCADH）和异戊酰辅酶A脱氢酶（IVDH）中，执行相同功能的相应Glu位于相邻螺旋G上的255位。因此，这些谷氨酸作用于从活性中心的两个相对区域接近的底物。我们已经通过实施Glu 376 Gly和Thr 255 Glu两个突变实现了MCADH中LCADH的拓扑结构。得到的嵌合酶，“中/长”链酰基辅酶A脱氢酶（MLCADH）具有与MCADH相似的20%的活性，与LCADH相似的25%的活性，其最佳底物分别为辛酰辅酶A和十二酰辅酶A。然而，MLCADH表现出与氧的再氧化速率增强，具有窄得多的底物链长度特异性，其与十二烷酰辅酶A达到峰值。这与LCADH的最大值相同，因此与天然MCADH（己酰/辛酰-CoA）的最大值显著偏移。LCADH和IVDH的推定的共同祖先具有两个Glu残基，在位置255和376处各一个。相应的MCADH突变体Thr 255 Glu（glu/glu-MCADH）与具有辛酰辅酶A的MCADH一样具有活性;然而，其活性/链长曲线窄得多。Glu作为H+提取碱基的拓扑结构似乎是决定酰基辅酶A脱氢酶链长特异性和反应性的重要因素。鉴于MLCADH的三维结构，讨论了这些作用的潜在机制，其在所附论文中给出[Lee等人（1996）Biochemistry 35，12412-12420]。
The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH). In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G. These glutamates thus act on substrate approaching from two opposite regions at the active center. We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu. The resulting chimeric enzyme, ''medium-/long'' chain acyl-CoA dehydrogenase (MLCADH) has similar to 20% of the activity of MCADH and similar to 25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively. MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA. This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA). The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376. The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower. The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases. The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper [Lee et al. (1996) Biochemistry 35, 12412-12420].