Joseph-McCarthy D, Petsko G A, Karplus M
Department of Chemistry, Harvard University, Cambridge, MA 02138, USA.
Protein Eng. 1995 Nov;8(11):1103-15. doi: 10.1093/protein/8.11.1103.
A minimum perturbation conformational search approach is used to model the structures of the yeast triosephosphate isomerase (TIM) single mutant in which the catalytic base Glu165 is changed to Asp, and the double mutant in which Glu165 is changed to Asp and Ser96 to Pro. In chicken TIM this double mutant is referred to as a pseudo-revertant because some of the catalytic activity lost due to the first mutation is regained when the second mutation occurs. Three minimum energy structures were calculated for the Asp165 conformation in the yeast TIM single mutant and another three for the double mutant. One of the calculated minimum energy conformations for Asp165 in the E165D structure agrees well with the X-ray structure. However, this conformation is not that of the lowest energy and is not one of the three most common conformers for Asp found by Ponder and Richards. This suggests that when an amino acid is introduced it may not be able to conform to the more general rules that apply to protein structures of evolutionary origin. While the van der Waals energy largely determines the allowed minima, the relative ranking of the final minima is determined by electrostatic effects and can therefore be affected by the inclusion of crystal waters in the calculation. When the E165D calculation is repeated with an active-site water molecule fixed in its E165D X-ray structure position, the relative ranking of the minima shifts and the X-ray conformation for Asp165 is the lowest interaction energy conformer. Two of the E165D calculated minimum energy structures are essentially identical to two of the S96P/E165D minima. All of the calculated minima for both the E165D and S96P/E165D mutants position the Asp side chain such that the anti-orbital, and not the more basic syn-orbital, of the carboxylate would be utilized for proton abstraction. This observation may explain why the chicken TIM S96P/E165D mutant, for which the X-ray structure indicates that the syn-orbital is used, is a pseudo-revertant while the yeast TIM double mutant is not; no X-ray structure is available for the latter. The multiplicity of minima found in the present analysis makes clear that predicting the exact orientation of a single side chain is not as simple as might be expected.
采用最小微扰构象搜索方法对酵母磷酸丙糖异构酶（TIM）单突变体（催化碱基Glu165突变为Asp）和双突变体（Glu165突变为Asp且Ser96突变为Pro）的结构进行建模。在鸡的TIM中，这种双突变体被称为假回复突变体，因为在发生第二次突变时，因第一次突变而丧失的部分催化活性得以恢复。对酵母TIM单突变体中Asp165构象计算了三个最低能量结构，对双突变体也计算了另外三个最低能量结构。在E165D结构中计算得到的Asp165的一个最低能量构象与X射线结构吻合良好。然而，这个构象并非能量最低的构象，也不是Ponder和Richards发现的Asp的三种最常见构象之一。这表明当引入一个氨基酸时，它可能无法遵循适用于进化起源蛋白质结构的更一般规则。虽然范德华能在很大程度上决定了允许的最小值，但最终最小值的相对排序由静电效应决定，因此在计算中包含结晶水可能会对其产生影响。当在E165D X射线结构位置固定一个活性位点水分子后重复E165D的计算时，最小值的相对排序发生变化，Asp165的X射线构象成为相互作用能最低的构象。计算得到的E165D的两个最低能量结构与S96P/E165D的两个最低能量结构基本相同。E165D和S96P/E165D突变体计算得到的所有最小值都将Asp侧链定位成这样，即羧酸盐的反式轨道而非更碱性的顺式轨道将用于质子提取。这一观察结果或许可以解释为什么鸡的TIM S96P/E165D突变体（其X射线结构表明使用的是顺式轨道）是假回复突变体，而酵母TIM双突变体不是；目前尚无后者的X射线结构。本分析中发现的多个最小值清楚地表明，预测单个侧链的确切取向并非如预期的那么简单。
