Crystal structure of the mutant yeast triosephosphate isomerase in which the catalytic base glutamic acid 165 is changed to aspartic acid.

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.