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 di ffraction at a nominal resolution of 2 Angstrom. For crystallization, the mutant enzyme was complexed with the tight-binding intermediate an alog, 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 aci d has increased the distance between the base and the intermediate ana log by 1 Angstrom. In addition, the catalytic base is oriented in the E165D structure so as to use the anti orbital of the carboxylate for p roton abstraction; in the structure of the wild-type enzyme, the syn o rbital 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 su bstrate. The structure of yeast E165D triosephosphate isomerase sugges ts that both distance and orientation factors contribute to the loss o f activity in the mutant enzyme and, therefore, that both factors cont ribute to the catalytic efficiency of the wild-type enzyme.