Abstract
Kinetic aspects of enzymatic reactions are described by equations based on the Michaelis-Menten theory for the initial stage. However, the kinetic parameters provide little information on the atomic mechanism of the reaction. In this study, we analyzed structures of glutamate dehydrogenase in the initial and steady stages of the reaction using cryoEM at near-atomic resolution. In the initial stage, four metastable conformations displayed different domain motions and cofactor/ligand association modes. The most striking finding was that the enzyme-cofactor-substrate complex, treated as a single state in the enzyme kinetic theory, comprised at least three different metastable conformations. In the steady stage, seven conformations, including derivatives from the four conformations in the initial stage, made the reaction pathway complicated. Based on the visualized conformations, we discussed stage-dependent pathways to illustrate the dynamics of the enzyme in action.