Enzymes involve many critical biological processes, and for some extends, the biological clock of a living cell is often regulated by enzymatic reactions. An enzymatic reaction involves active substrate-enzyme complex formation, chemical transformation, and product releasing, as we know of the Mechalis-Menten mechanism. Enzymes can change the biological reaction pathways and accelerate the reaction rate by thousands and even millions of times. It is the enzyme-substrate interaction and complex formation that play a critical role in defining the enzymatic reaction landscape, including reaction potential surface, transition states of chemical transformation, and oscillatory reaction pathways. Subtle conformational changes play a crucial role in enzyme functions, and these protein conformations are highly dynamic rather than being static. Using only a static structural characterization, from an ensemble-averaged measurement at equilibrium is often inadequate in predicting dynamic conformations and understanding correlated enzyme functions in real time involving in nonequilibrium, multiple-step, multiple-conformation complex chemical interactions and transformations. Single-molecule assays have revealed static [1-5] and dynamic [3-5] disorders in enzymatic reactions by probing co-enzyme redox state turnovers [3] and enzymatic reaction product formation in real time [4,5]. Static and dynamic disorders [6-10] are, respectively, the static rate inhomogeneities between molecules and the dynamic rate fluctuations for individual molecules. Dynamic disorder, which is not distinguishable from static disorder in an ensemble-averaged measurement, has been attributed to protein conformational fluctuations [3-5,11]. The protein conformational motions at the enzyme active site, which include enzyme-substrate complex formation, enzymatic reaction turnovers, and product releasing, are mostly responsible for the inhomogeneities in enzymatic reactions [3-5]. Consequently, direct observations of conformational changes along enzymatic reaction coordinates are often crucial for understanding inhomogeneities in enzymatic reaction systems [12]. We have applied single-molecule spectroscopy and imaging to study complex enzymatic reaction dynamics and the enzyme conformational changes, focusing on the T4 lysozyme enzymatic hydrolyzation of the polysaccharide walls of Escherichia, coli B cells. By attaching a donor-acceptor pair of dye molecules site-specifically to noninterfering sites on the enzyme, we were able to measure the hinge-bending conformational motions of the active enzyme by monitoring the donor acceptor emission intensity as a function of time. We have also explored a combined approach, applying molecular dynamics (MD) simulation and a random-walk model based on the single-molecule experimental data. Using this approach, we analyzed enzyme substrate complex formation dynamics to reveal (1) multiple intermediate conformational states, (2) oscillatory conformational motions, and (3) a conformational memory effect in the chemical reaction process [13]. Moving forward to study enzymatic dynamics and enzyme conformational dynamics in living cells, we have developed a single-molecule enzyme delivery approach to place an enzyme specifically to an enzymatic reaction site on a cell membrane.
