| Enzymes are biological catalysts vital to life processes and have been the subject of century-long investigations. The classic Michaelis-Menten equation provides a highly satisfactory description of enzymatic kinetics for large ensembles of enzyme molecules. Here, we present a single-molecule assay that records enzymatic trajectories of individual enzyme molecules. The study of single enzyme molecules has made it possible to address a number of intriguing questions: How are single-molecule enzymatic measurements, which record stochastic waiting times of enzymatic reactions, reconciled with ensemble Michaelis-Menten kinetics? What new information is available from single-molecule experiments? In particular, what are the magnitude and time scales of the enzymatic rate fluctuations and what implication, if any, do these fluctuations have on cellular processes? We have developed a novel fluorescent product assay to experimentally test the validity of the fundamental Michaelis-Menten equation at the single molecule level. In order to acquire the statistics necessary for reliable data analyses, we conduct experiments on beta-galactosidase with a fluorogenic substrate that allows us to extend observation trajectories to twenty thousand total turnovers. We also present an assay to study the turnover dynamics of individual EcoRV restriction endonuclease enzymes. The broad time scales of enzymatic rate fluctuations both for beta-galactosidase as well as for EcoRV, which are uncovered by analyses of long turnover time traces, emphasizes the fact that an enzyme molecule is an ever fluctuating dynamic entity during catalysis. Our analysis has not only confirmed the validity of the single-molecule MM-equation but also has revealed a memory effect that lasts for decades of time-scales. Our study probes the importance of turnover fluctuations on systems containing a few beta-galactosidase enzyme molecules. In particular, we find that if a system contains only a small number of enzyme molecules, as is often the case in a living cell, the enzymatic fluctuations may be readily manifested. |
