Several hundred single GUS molecules were separated in large arrays of 62 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis—Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS.
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Get e-Alerts Abstract This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. In both cases. This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.
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Role of substrate unbinding in Michaelis–Menten enzymatic reactions
Get e-Alerts Abstract This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. In both cases. This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening. Cited By This article is cited by publications.
Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited
With the advent of high-resolution methods of single-molecule spectroscopy, it is now possible to directly observe and manipulate the behavior of individual enzymes in the course of a chemical reaction. Chemistry at the single-molecule level is, however, inherently stochastic and, at times, extremely unintuitive. In this paper, we explain why, and under what circumstances, an increase in the rate at which an enzyme unproductively departs from a bound substrate will—unexpectedly—lead to an acceleration in the rate of product formation. The far-reaching implications of this effect are discussed. Keywords: single enzyme, enzyme kinetics, renewal theory Abstract The Michaelis—Menten equation provides a hundred-year-old prediction by which any increase in the rate of substrate unbinding will decrease the rate of enzymatic turnover.
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