Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited

PHYS 159

Brian P. English, benglish@fas.harvard.edu1, Wei Min, weimin@fas.harvard.edu1, A. M. van Oijen, vanoijen@fas.harvard.edu1, Samuel Kou, kou@stat.harvard.edu2, and X. Sunney Xie, xie@chemistry.harvard.edu1. (1) Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, (2) Department of Statistics, Harvard University, 1 OXFORD ST, Cambridge, MA 02138
The Michaelis-Menten equation accurately describes the bulk behavior of thousands of enzymes and lays the foundation of modern biochemistry. We set out to test if the classic Michaelis-Menten equation holds also for individual enzyme molecules. A novel single-molecule assay is developed to monitor long time traces of enzymatic turnovers for individual β-galactosidase molecules by detecting one fluorescent product molecule at a time. The continuous replenishing of fluorescent signal considerably extends the observable timescale that previously was limited by photobleaching. A molecular memory phenomenon arises at high substrate concentrations, characterized by clusters of turnover events separated by periods of low activity. Such memory lasts for decades of time scales ranging from milliseconds to seconds due to interconverting conformers with broadly distributed lifetimes. We prove that the Michaelis-Menten equation still holds even for a fluctuating single enzyme, but bears a different microscopic interpretation.