Single-Molecule Enzymatic Conformational Dynamics: Spilling Out the Product Molecules

Abstract

Product releasing is an essential step of an enzymatic reaction, and a mechanistic understanding primarily depends on the active-site conformational changes and molecular interactions that are involved in this step of the enzymatic reaction. Here we report our work on the enzymatic product releasing dynamics and mechanism of an enzyme, horseradish peroxidase (HRP), using combined single-molecule time-resolved fluorescence intensity, anisotropy, and lifetime measurements. Our results have shown a wide distribution of the multiple conformational states involved in active-site interacting with the product molecules during the product releasing. We have identified that there is a significant pathway in which the product molecules are spilled out from the enzymatic active site, driven by a squeezing effect from a tight active-site conformational state, although the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway. Our study provides new insight into the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined time-resolved single-molecule spectroscopic measurements and analyses.

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

“…However, the complexity is narrowed or even eliminated when the enzyme is deformed or unfolded under magnetic pulling force. microseconds it provides a direct readout of enzymatic reactions with single turnover resolution (5,8,9,(22)(23)(24). Additionally, fluorescent product molecules are produced continuously in turnover cycles and this eliminates the negative impact of photobleaching, and thereby long time trajectories from a single enzyme can be recorded (8,9).…”

“…microseconds it provides a direct readout of enzymatic reactions with single turnover resolution (5,8,9,(22)(23)(24). Additionally, fluorescent product molecules are produced continuously in turnover cycles and this eliminates the negative impact of photobleaching, and thereby long time trajectories from a single enzyme can be recorded (8,9). In this work, we extend the SM fluorogenic assay and imaging analysis to a new dimension by combing the total internal reflection fluorescence microscopy (TIRFM)-guided confocal time-resolved SM photon time-stamping spectroscopic approach and the SM fluorogenic assay combined with magnetic tweezers force manipulation of molecular conformations: We used the nascent formed fluorescent product as an in situ probe to study the active site conformational fluctuation dynamics during the enzymatic reaction from the moment the product incipiently formed to the releasing of the fluorescent product from the active site (9,20,25).…”

“…Additionally, fluorescent product molecules are produced continuously in turnover cycles and this eliminates the negative impact of photobleaching, and thereby long time trajectories from a single enzyme can be recorded (8,9). In this work, we extend the SM fluorogenic assay and imaging analysis to a new dimension by combing the total internal reflection fluorescence microscopy (TIRFM)-guided confocal time-resolved SM photon time-stamping spectroscopic approach and the SM fluorogenic assay combined with magnetic tweezers force manipulation of molecular conformations: We used the nascent formed fluorescent product as an in situ probe to study the active site conformational fluctuation dynamics during the enzymatic reaction from the moment the product incipiently formed to the releasing of the fluorescent product from the active site (9,20,25). It is a conceptual and technical advancement to use a nascent-formed fluorogenic product molecule serving as a probe for enzymatic reaction active site with perfectly fitted position with the critical molecular interactions without perturbing the active site as in the case of site-specific labeling.…”

“…In recent years, it has been widely identified that conformational dynamics plays a crucial role in regulating enzymatic reactions (2,(4)(5)(6)(7). The advent of sitespecific mutagenesis, single-molecule (SM) spectroscopic technique, and molecular dynamics simulations have demonstrated complex dynamics involving multiple conformational states during a catalytic cycle (4,5,(7)(8)(9)(10)(11)(12)(13). Fundamentally, not only the enzymatic active site conformational dynamics but also the overall conformational fluctuation dynamics of the protein matrix, providing the required entropy, enthalpy, and corresponding energy landscape, has a profound impact on an enzyme to ultimately possess the power of enhancing reaction rates.…”

“…Few studies have tried to address these questions and related effects on other macroscopic parameters directly through experimental approaches (9,(15)(16)(17)(18)(19). We recently reported that horseradish peroxidase (HRP) can involve multiple intermediate conformational states and pathways during product releasing, and a significant pathway is that the product molecules are spilled out from the enzymatic reaction site; however, releasing the product molecules through a loosely bound enzyme-product state and an open active site remain parallel product releasing pathways (9). Further, very recently our group has reported that HRP retains a significant amount of its activity even under conformational perturbations (20).…”