Abstract
Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.
Highlights
Enzymes are omnipresent catalysts of biochemical reactions
Fluorescence microscopy has become an essential method for the non-invasive interrogation of biomolecules, invigorated by new methods to increase the optical resolution of microscopy beyond the diffraction limit of light
This review focuses on the second type of single enzyme molecule experiments that can investigate the catalytic activity of individual enzyme molecules without the need for detecting single fluorophore molecules
Summary
Enzymes are omnipresent catalysts of biochemical reactions. Detailed studies of enzymes and their catalytic activity have provided us with a global understanding of enzyme structure and functionality. Different conformational states of individual enzyme molecules entail dynamic fluctuations such as varying substrate turnover rates over time (dynamic heterogeneity) [7,8,9,10] or broad distributions of catalytic rates within an enzyme population (static heterogeneity) [11,12,13]. The reaction rate of an individual enzyme was evaluated from the inverse of the mean waiting time 〈 〉 between two successive turnover events In this case, the Michaelis-Menten equation was reformulated to:. Technologies for single molecule enzymology have disclosed fascinating kinetic details They have uncovered enzymatic subpopulations hidden in bulk-phase experiments and enabled the assessment of cooperativity in oligomeric enzymes such as β-galactosidase [20]. The development of more sensitive detection methods and new single molecule technologies has greatly extended our understanding of the fundamental biochemical processes of life
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