Distinct and Long-Lived Activity States of Single Enzyme Molecules

Abstract

Individual enzyme molecules have been observed to possess discrete and different turnover rates due to the presence of long-lived activity states. These stable activity states are thought to result from different molecular conformations or post-translational modifications. The distributions in kinetic activity observed in previous studies were obtained from small numbers of single enzyme molecules. Due to this limitation, it has not been possible to fully characterize the different kinetic and equilibrium binding parameters of single enzyme molecules. In this paper, we analyze hundreds of single beta-galactosidase molecules simultaneously; using a high-density array of 50,000 fL-reaction chambers, we confirm the presence of long-lived kinetic states within a population of enzyme molecules. Our analysis has isolated the source of kinetic variability to kcat. The results explain the kinetic variability within enzyme molecule populations and offer a deeper understanding of the unique properties of single enzyme molecules. Gaining a more fundamental understanding of how individual enzyme molecules work within a population should provide insight into how they affect downstream biochemical processes. If the results reported here can be generalized to other enzymes, then the stochastic nature of individual enzyme molecule kinetics should have a substantial impact on the overall metabolic activity within a cell.

Single-molecule approaches to characterizing kinetics of biomolecular interactions

“…Different members of a seemingly homogeneous collection of molecules may exhibit different binding kinetics (so-called static disorder), or individual members of a population may exhibit binding kinetics that change over time (dynamic disorder) [11][12][13][14][15][16]. Characterizing these different phenomena and correlating them with information on structural conformation will increase our understanding of the relationship between activity and structure, an important factor in both binding and enzymatic activity [14,[17][18][19][20][21][22].…”

Single molecule kinetics of horseradish peroxidase exposed in large arrays of femtoliter-sized fused silica chambers

“…Strong excitation light, however, led to photooxidation of Amplex Red, 23 which increased the uorescence background, and photobleaching of the product resorun. 7,13 Here, we have optimized the optical path as well as the detection system to allow for employing milder excitation conditions. A femtoliter array etched into the surface of a fused silica slide was directly monitored under a high resolution objective.…”

Stochastic mapping of the Michaelis-Menten mechanism

“…[3][4][5] With the advance of detection technology, it is now possible to study the activity of a single enzyme. [6][7][8][9][10][11][12][13] For example, the activity of a single cholesterol oxidase molecule could be observed through fluorescent microscopy by detecting the emission from the enzyme's fluorescent active site, flavin adenine dinucleotide. 6 In addition to following singlemolecule events, these measurements also gave evidence of slow fluctuations in protein conformation.…”

“…12,22 Recent advance in the stochastic modeling of absolute asymmetric reactions shows that it is often possible to obtain meaningful analytical solutions for systems exhibiting moderate levels of complexity, and give reasonably complete general mathematical descriptions of those chemical schemes. [29][30][31] This paper attempts to carry out a similar analysis for the Michaelis-Menten mechanism.…”