Conformational flexibility of proteins provides enzymes with high catalytic activity. Although the conformational flexibility is known to be pivotal for the ligand binding and release, its role in the chemical reaction process of the reactive substrate remains unclear. We determined a transition state of an enzymatic reaction in a psychrophilic a-amylase by a hybrid molecular simulation that allows one to identify the optimal chemical state in an extensive conformational ensemble of protein. The molecular simulation uncovered that formation of the reaction transition state accompanies a large and slow movement of a loop adjacent to the catalytic site. Free energy calculations revealed that, although catalytic electrostatic potentials on the reactive moiety are formed by local and fast reorganization around the catalytic site, reorganization of the large and slow movement of the loop significantly contributes to reduction of the free energy barrier by stabilizing the local reorganization.