PNNL

Abstract

Enzymatic turnovers of single cholesterol oxidase molecules are observed in real time by monitoring the emission from the enzyme's fluorescent active site, flavin adenine dinucleotide (FAD). Although chemical kinetics, the Michaelis-Menten mechanism in particular, holds as a good approximation, statistical analyses of single-molecule trajectories reveal fluctuations in the rate of the activation step in the Michaelis-Menten mechanism. There exists a memory effect: an enzymatic turnover is not independent of its previous turnovers. This non-Markovian behavior, otherwise hidden in ensemble-averaged meausrements, is attributed to slow fluctuations of protein conformations. Static heterogeneity and dynamical variation of reaction rates, essentially indistinguishable in ensemble-averaged experiments, can now be determined separately by the real-time single-molecule approach.