Abstract
To understand the catalytic mechanism of an enzyme, it is crucial to determine the crystallographic structures corresponding to the individual reaction steps. Here we report two crystal structures of enzyme-substrates complexes prior to reaction initiation : tropinone reductase-II (TR-II) -NADPH and TR-II-NADPH-tropinone complexes, determined from the identical crystals. A combination of two kinetic crystallographic techniques, a continuous flow of the substrates and Laue diffraction measurement, enabled us to capture the transit structures prior to the reaction proceeding. A structure comparison of enzyme-substrates complex elucidated in this study with the enzyme-products complex in our previous study indicates that one of the substrates, tropinone, is rotated relative to the product so as to make the spatial organization in the active site favorable for the reaction to proceed. Side chains of the residues in the active site also alter their conformations to keep the complementarily of the space for the substrate or the product and to assist the rotational movement.
