Although enzymatic reactions are traditionally studied at the ensemble level, the inhomogeneities of the reaction rate, the correlated enzyme conformational motions, and the non-synchronized nature of these reactions make it extremely difficult to obtain stepwise mechanistic and dynamic information from such studies. Presumably, the protein conformational motions at the active site of the enzyme are most critically responsible for the inhomogeneities in the enzymatic reactions. It will be critical in understanding the mechanisms and dynamics of intrinsic inhomogeneous and non-synchronizable enzymatic reactions to obtain real-time observations showing the correlation of single-molecule enzymatic reactions with unambiguously assigned protein conformational motions. The long-term goal of this project is to extend our molecule-level understanding of protein conformational fluctuation and relaxation dynamics and their impact on enzymatic reaction dynamics. Non-Arrhenius and non-Markovian dynamics, historesis, cooperativity, and memory effects of enzymatic reactions are widely observed but are insufficiently understood due to the nature of the inhomogeneity in an ensemble-averaged measurement. Applying single-molecule spectroscopy, we were able to probe protein conformational motions of a single enzyme (T4 lysozyme, an 18.6-kDa, single-subunit, and 164-amino acid enzyme comprising two domains connected by an α-helix) during hydrolysis of the polysaccharide walls of Escherichia coli B cells. By attaching a donor-acceptor (tetramethylrhodamine-Texas Red) pair of dye molecules site-specifically to non-interfering sites on the enzyme, we measured the dynamics of the hinge-bending motions of the enzyme under enzymatic turnovers by monitoring the donor-acceptor emission intensity changes due to single-pair fluorescence resonance energy transfer (spFRET). The overall enzymatic reaction rate constants were found to vary widely from molecule to molecule under the same physiological condition. The enzyme’s searching for reactive sites in the substrate was found to account for most of this inhomogeneity. By applying a molecular dynamics (MD) simulation and a random-walk model to analyze the enzyme-substrate complex formation dynamics, we have revealed multiple intermediate conformational states in the chemical reaction process. This approach provides information on the microscopic conformational change mean drifting velocity, diffusion coefficient, friction coefficient, energy consumed by friction along the reaction coordinate, and the energy landscape. This information cannot be obtained by an ensemble-averaged experiment, but is obtainable by a single-molecule approach. Our combined approach achieved the simultaneous measurement of enzyme conformational motion and enzymatic turnovers and demonstrates the potential of single-molecule studies in understanding non-synchronizable, multi-step reaction dynamics and mechanisms, eventually, in living cells.
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