Revealing Multiple Pathways in T4 Lysozyme Substep Conformational Motions by Single-Molecule Enzymology and Modeling.

Enzyme conformational dynamics play crucial roles in catalytic functions. Obtaining molecular level insights into the conformational transition dynamics of enzyme-substrate complex from the inactive state to the active state is fundamental for understanding enzymatic function and dynamics. Here, we report our progress on the real-time conformational transition dynamics of T4 lysozyme under enzymatic reactions using single-molecule fluorescence resonance energy transfer. The time duration in forming the active enzyme-substrate complex state (ES*) shows distinctive Poisson and non-Poisson statistics, including exponential and nonexponential, convoluted Poisson distributions, and Gaussian-like distributions. These complex dynamic behaviors of T4 lysozyme are in excellent agreement with a Markov dynamic simulation and a transition steps modeling. Specifically, we are able to obtain mechanistic understandings: (1) Transiting from enzyme (E) to ES*, T4 lysozyme hinge-bending conformational changes undergo multiple steps following multiple pathways. In the case of shortest pathway, this transition only requires one elementary transition or reaction step. (2) Substep conformational motions, associating with multiple nuclear coordinates and a common projected FRET-sensitive nuclear coordinate, can give rise to multiple conformational intermediate states. (3) The consequence of the multiple pathways, intermediate states, and nuclear coordinates is the time bunching effect; i.e., time durations of conformational changes tend to bunch in a narrowly distributed time window. The physical picture of multiple intermediate states along with bunching effect suggests that the conformational dynamics of T4 lysozyme shows a complementary characteristic behavior of convoluted enzyme conformation selection and induced-fit dynamics driven by substrate-enzyme interactions.