Nandy A, Kieweg V, Kräutle F G, Vock P, Küchler B, Bross P, Kim J J, Rasched I, Ghisla S
Faculty of Biology, University of Konstanz, Germany.
Biochemistry. 1996 Sep 24;35(38):12402-11. doi: 10.1021/bi960785e.
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 approximately 20% of the activity of MCADH and approximately 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].
催化过程中通过夺取底物α-氢作为H⁺从而启动催化作用的关键谷氨酸残基，位于中链酰基辅酶A脱氢酶（MCADH）中JK环的376位（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谷氨酸位于相邻螺旋G的255位。因此，这些谷氨酸作用于从活性中心两个相对区域接近的底物。我们通过进行Glu376Gly和Thr255Glu这两个突变，在MCADH中实现了LCADH的拓扑结构。所得的嵌合酶“中/长”链酰基辅酶A脱氢酶（MLCADH），分别以其最佳底物辛酰辅酶A和十二烷酰辅酶A，具有约20%的MCADH活性和约25%的LCADH活性。然而，MLCADH表现出与氧气再氧化的速率增强，但其底物链长特异性要窄得多，以十二烷酰辅酶A时达到峰值。这与LCADH的最大值相同，因此与天然MCADH（己酰/辛酰辅酶A）有显著差异。推测的LCADH和IVDH的共同祖先有两个谷氨酸残基，分别位于255位和376位。相应的MCADH突变体Thr255Glu（glu/glu-MCADH），以辛酰辅酶A为底物时与MCADH活性相当；然而，其活性/链长曲线要窄得多。谷氨酸作为夺取H⁺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一个重要因素。鉴于随附论文[Lee等人（1996年）《生物化学》35卷，12412 - 12420页]中呈现的MLCADH的三维结构，讨论了这些效应背后的机制。
