一个晶体比较突变glyceraldehyde-3-phosphate脱氢酶从芽孢杆菌stearothermophilus包裹着NAD +或辅酶ii +。
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Didierjean C, Rahuel-Clermont年代,Vitoux B, Dideberg O, Branlant G,奥布里
一个晶体比较突变glyceraldehyde-3-phosphate脱氢酶从芽孢杆菌stearothermophilus包裹着NAD +或辅酶ii +。
J杂志。1997年5月16日,268 (4):739 - 59。
- PubMed ID
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9175858 (在PubMed]
- 文摘
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介绍了突变的胞质glyceraldehyde-3-phosphate脱氢酶(GAPDH)杆菌stearothermophilus为了对NAD的代数余子式从特异性选择性转化为辅酶ii。台球突变体,五个突变(精英语言、T34G D35G, L187A, P188S)的基础上选择一个序列比对与NADP-dependent叶绿素GAPDHs。D32G-S突变体,两个上面提到的五个突变(L187A P188S)已经与另一个设计静电结合使用注意事项(D32G)。两个突变体表现出dual-cofactor选择性优势的NAD(台球)或辅酶ii (D32G-S)。为了分析cofactor-binding网站可塑性在分子水平上,这些突变体已经解决了晶体结构,当包裹着NAD + (D32G-Sn、分辨率2.5 R = 13.9%;B-Sn, 2.45, 19.3%)或辅酶ii + (D32G-Sp, 2.2一个,19.2%;B-Sp, 2.5, 14.4%)。四个精致的模型非常类似于野生型GAPDH和预期更多的相似比apo形式整体形式。台球突变,野生型低亲和力辅酶ii +似乎基本上保留因为排斥静电联系人之间的额外的2的磷酸和残留D32不变羧基。这样一个antideterminant公认的有吸引力的互动效应是不补偿的预期出现的新推出侧链。 In this mutant, recognition of NAD+ is slightly affected with respect to that known on the wild-type, because mutations only weakly destabilize hydrogen bonds and van der Waals contacts originally present in the natural enzyme. Thus, the B-S mutant does not mimic efficiently the chloroplastic GAPDHs, and long-range and/or second-layer effects, not easily predictable from visual inspection of three-dimensional structures, need to be taken into account for designing a true "chloroplastic-like" mutant of cytosolic GAPDH. In the case of the D32G-S mutant, the dissociation constants for NAD+ and NADP+ are practically reversed with respect to those of the wild-type. The strong alteration of the affinity for NAD+ obviously proceeds from the suppression of the two wild-type hydrogen bonds between the adenosine 2'- and 3'-hydroxyl positions and the D32 carboxylate group. As expected, the efficient recognition of NADP+ is partly promoted by the removal of intra-subunit electrostatic repulsion (D32G) and inter-subunit steric hindrance (L187A, P188S). Another interesting feature of the reshaped NADP+-binding site is provided by the local stabilization of the extra 2'-phosphate which forms a hydrogen bond with the side-chain hydroxyl group of the newly-introduced S188. When compared to the presently known natural NADP-binding clefts, this result clearly demonstrates that an absolute need for a salt-bridge involving the 2'-phosphate is not required to switch the cofactor selectivity from NAD to NADP. In fact, as it is the case in this mutant, only a moderately polar hydrogen bond can be sufficient to make the extra 2'-phosphate of NADP+ well recognized by a protein environment.