Enzymes are biological catalysts vital to life processes and have attracted century-long investigation. The classic Michaelis-Menten mechanism provides a highly satisfactory description of catalytic activities for large ensembles of enzyme molecules. Here we tested the Michaelis-Menten equation at the single-molecule level. We monitored long time traces of enzymatic turnovers for individual β-galactosidase molecules by detecting one fluorescent product at a time. 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 timescales ranging from milliseconds to seconds owing to the presence of interconverting conformers with broadly distributed lifetimes. We proved that the Michaelis-Menten equation still holds even for a fluctuating single enzyme, but bears a different microscopic interpretation.
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We thank E.J. Sánchez for developing the scanning software and P. Blainey for his help with Matlab simulations. This work was funded by a US National Institutes of Health (NIH) R01 grant and recently by the NIH Director's Pioneer Award to X.S.X. B.P.E. is supported by an NIH Training Grant. K.T.L is supported by the Post-doctoral Fellowship Program of Korea Science & Engineering Foundation. A.M.v.O. acknowledges financial support from the Niels Stensen Foundation. S.C.K. acknowledges support from an NSF grant and an NSF CAREER award.
The authors declare no competing financial interests.
Effect of surface immobilization on enzymatic activity. (PDF 77 kb)
Loss of enzymatic activity due to tetrameter dissociation. (PDF 78 kb)
Autohydrolysis rate. (PDF 94 kb)
Effectiveness of the bleaching scheme. (PDF 78 kb)
Intensity histogram. (PDF 70 kb)
Autocorrelation of k(t). (PDF 546 kb)
Monte-Carlo simulations. (PDF 120 kb)
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English, B., Min, W., van Oijen, A. et al. Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited. Nat Chem Biol 2, 87–94 (2006). https://doi.org/10.1038/nchembio759
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