We demonstrate the use of single-molecule spectroscopy to study enzyme conformational motions of T4 lysozyme under hydrolysis reaction of the polysaccharide walls of E. coli B cells. By attaching a donor-acceptor pair of dye molecules site-specifically to noninterfering sites on the enzyme, the hinge-bending motions of the enzyme are measured by monitoring the donor-acceptor emission intensity as a function of time. The overall enzymatic reaction rate constants are found to vary widely from molecule to molecule. The dominant contribution to this static inhomogeneity is attributed to enzyme searching for reactive sites on the substrate. We have also applied molecular dynamics simulation and a random-walk model to analyze the enzyme-substrate complex formation dynamics, revealing multiple intermediate conformational states in the chemical reaction process. This approach provides information about the microscopic conformational change drifting velocity, diffusion coefficient, friction coefficient, energy consumed by friction along the reaction coordinate, and energy landscape.