Joseph-McCarthy D, Rost L E, Komives E A, Petsko G A
Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110.
Biochemistry. 1994 Mar 15;33(10):2824-9. doi: 10.1021/bi00176a011.
The three-dimensional structure of the E165D mutant of the glycolytic enzyme yeast triosephosphate isomerase has been determined by X-ray diffraction at a nominal resolution of 2 A. For crystallization, the mutant enzyme was complexed with the tight-binding intermediate analog, phosphoglycolohydroxamate. Comparison with the structure of the wild-type enzyme reveals that, as originally intended, replacement of the catalytic base Glu-165 with the shorter side chain of aspartic acid has increased the distance between the base and the intermediate analog by 1 A. In addition, the catalytic base is oriented in the E165D structure so as to use the anti orbital of the carboxylate for proton abstraction; in the structure of the wild-type enzyme, the syn orbital is oriented for this purpose. It has been hypothesized that the 1000-fold loss in catalytic activity for this mutant triosephosphate isomerase is due either to the use of the less basic anti orbital for proton transfer or to the greater distance between the base and the substrate. The structure of yeast E165D triosephosphate isomerase suggests that both distance and orientation factors contribute to the loss of activity in the mutant enzyme and, therefore, that both factors contribute to the catalytic efficiency of the wild-type enzyme.
已通过X射线衍射在标称分辨率为2埃的条件下测定了糖酵解酶酵母磷酸丙糖异构酶E165D突变体的三维结构。为了进行结晶，将突变酶与紧密结合的中间类似物磷酸甘油异羟肟酸复合。与野生型酶的结构比较表明，正如最初预期的那样，用天冬氨酸较短的侧链取代催化碱基Glu-165，使碱基与中间类似物之间的距离增加了1埃。此外，催化碱基在E165D结构中的取向是利用羧酸盐的反式轨道进行质子提取；在野生型酶的结构中，为此目的取向的是顺式轨道。据推测，这种突变的磷酸丙糖异构酶催化活性降低1000倍，要么是由于使用了碱性较弱的反式轨道进行质子转移，要么是由于碱基与底物之间的距离更大。酵母E165D磷酸丙糖异构酶的结构表明，距离和取向因素都导致了突变酶活性的丧失，因此，这两个因素都对野生型酶的催化效率有贡献。
