Komives E A, Lougheed J C, Liu K, Sugio S, Zhang Z, Petsko G A, Ringe D
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0601, USA.
Biochemistry. 1995 Oct 17;34(41):13612-21. doi: 10.1021/bi00041a041.
The structural basis for the improvement in catalytic efficiency of the mutant E165D chicken triosephosphate isomerase by the secondary mutation, S96P, has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. All X-ray structures were of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog, with the isomerase, and each was solved to a resolution of 1.9 A. Comparison of the structure of the double mutant, E165D.S96P, with that of the single mutant, E165D, as well as with the wild-type isomerase shows only insignificant differences in the positions of the side chains in all of the mutants when compared with the wild-type isomerase, except that in both the E165D and E165D.S96P mutants, the aspartate side chain was approximately 0.7 A further away from the substrate analog than the glutamate side chain. Significant differences were observed in the crystal structure of the E165D.S96P double mutant in the positions of ordered water molecules bound at the active site. The loss of two water molecules located near the side chain at position 165 was observed in isomerases containing the S96P mutation. The resulting increase in hydrophobicity of the pocket probably causes an increase in the pKa of the catalytic base, D165, thereby improving its basicity. A new ordered water molecule was observed underneath the bound PGH in the E165D.S96P structure, which likely decreases the pKa's of the substrate protons, thereby increasing their acidity. An enzyme derived carbonyl stretch at 1746 cm-1 that is only observed in the IR spectrum of the E165D.S96P double mutant isomerase with bound substrates has been assigned to a stable ground state protonated D165-enediol(ate) intermediate complex. Thus, the gain in activity resulting from the S96P second site change probably results from a combination of improving the basicity of the enzyme, improving the acidity of the substrate protons, and stabilization of a reaction intermediate. All three of these effects seem to be caused by changes in bound water molecules.
通过结合X射线晶体学和傅里叶变换红外光谱，分析了二级突变S96P对突变体E165D鸡磷酸丙糖异构酶催化效率提高的结构基础。所有X射线结构均为磷酸甘油异羟肟酸（PGH，一种中间类似物）与异构酶的复合物，且每种结构的分辨率均达到1.9 Å。将双突变体E165D.S96P的结构与单突变体E165D以及野生型异构酶的结构进行比较，结果表明，与野生型异构酶相比，所有突变体侧链位置的差异均不显著，只是在E165D和E165D.S96P突变体中，天冬氨酸侧链与底物类似物的距离比谷氨酸侧链大约远0.7 Å。在E165D.S96P双突变体的晶体结构中，观察到活性位点结合的有序水分子位置存在显著差异。在含有S96P突变的异构酶中，观察到位于165位侧链附近的两个水分子缺失。口袋疏水性的增加可能导致催化碱基D165的pKa升高，从而提高其碱性。在E165D.S96P结构中，在结合的PGH下方观察到一个新的有序水分子，这可能会降低底物质子的pKa，从而增加其酸度。仅在结合底物的E165D.S96P双突变体异构酶的红外光谱中观察到的1746 cm-1处的酶衍生羰基伸缩振动，已被指定为稳定的基态质子化D165-烯二醇（盐）中间复合物。因此，S96P第二位点变化导致的活性增加可能是由于提高了酶的碱性、提高了底物质子的酸度以及稳定了反应中间体这三种效应共同作用的结果。所有这三种效应似乎都是由结合水分子的变化引起的。
