Limits...
Single-molecule enzymatic conformational dynamics: spilling out the product molecules.

Zheng D, Lu HP - J Phys Chem B (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University , Bowling Green, Ohio 43403, United States.

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.

Show MeSH

Related in: MedlinePlus

Single-molecule fluorescence lifetime and anisotropydistributionsof the rising edges and falling edges of single HRP-catalyzed oxidationof amplex red reaction fluorescence turnover events. (A1) The lifetimedistribution of rising edges with mean at 3.75 ns and standard deviation1.26 ns. (A2) The anisotropy distribution of rising edges with meanat 0.17 and standard deviation 0.14 ns. (B1) The lifetime distributionof falling edges with mean at 3.37 ns and standard deviation 1.3.(B2) The anisotropy distribution of falling edges with mean at 0.15and standard deviation 0.16.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126733&req=5

fig6: Single-molecule fluorescence lifetime and anisotropydistributionsof the rising edges and falling edges of single HRP-catalyzed oxidationof amplex red reaction fluorescence turnover events. (A1) The lifetimedistribution of rising edges with mean at 3.75 ns and standard deviation1.26 ns. (A2) The anisotropy distribution of rising edges with meanat 0.17 and standard deviation 0.14 ns. (B1) The lifetime distributionof falling edges with mean at 3.37 ns and standard deviation 1.3.(B2) The anisotropy distribution of falling edges with mean at 0.15and standard deviation 0.16.

Mentions: To further identify the conformational dynamics of the productreleasing from the enzyme active site, we have analyzed the distributionsof the fluorescence lifetime and anisotropy at the rising edges andfalling edges of about 211 fluorescence bursting spikes from the trajectoriesshown in Figure 2. Here, the signal risingedge corresponds to the fluorescent product generated at a single-moleculereactant-to-product turnover, and the signal falling edge correspondsto the product releasing from the active site into the solution anddiffusing away from the confocal volume. Figure 6 shows the distributions of the lifetimes and anisotropies of theproducts from the rising edges and the falling edges of each fluorescencespike. Comparing the lifetime distributions from the rising and fallingedges of the fluorescence spikes (Figure 6 A1and Figure 6 B1), we notice that the mean lifetimeof the falling part is about 0.38 ns shorter than that of the risingpart, while the anisotropy distributions are almost the same. On thebasis of the influence of the quenching effect and local dielectricconstant on the fluorescence lifetime of the nascent product as discussedabove, the most possible explanation for the shorter lifetime of thesingle resorufin molecules at the falling edges is that the productmolecules are buried in tight states compared to the rising edge.


Single-molecule enzymatic conformational dynamics: spilling out the product molecules.

Zheng D, Lu HP - J Phys Chem B (2014)

Single-molecule fluorescence lifetime and anisotropydistributionsof the rising edges and falling edges of single HRP-catalyzed oxidationof amplex red reaction fluorescence turnover events. (A1) The lifetimedistribution of rising edges with mean at 3.75 ns and standard deviation1.26 ns. (A2) The anisotropy distribution of rising edges with meanat 0.17 and standard deviation 0.14 ns. (B1) The lifetime distributionof falling edges with mean at 3.37 ns and standard deviation 1.3.(B2) The anisotropy distribution of falling edges with mean at 0.15and standard deviation 0.16.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126733&req=5

fig6: Single-molecule fluorescence lifetime and anisotropydistributionsof the rising edges and falling edges of single HRP-catalyzed oxidationof amplex red reaction fluorescence turnover events. (A1) The lifetimedistribution of rising edges with mean at 3.75 ns and standard deviation1.26 ns. (A2) The anisotropy distribution of rising edges with meanat 0.17 and standard deviation 0.14 ns. (B1) The lifetime distributionof falling edges with mean at 3.37 ns and standard deviation 1.3.(B2) The anisotropy distribution of falling edges with mean at 0.15and standard deviation 0.16.
Mentions: To further identify the conformational dynamics of the productreleasing from the enzyme active site, we have analyzed the distributionsof the fluorescence lifetime and anisotropy at the rising edges andfalling edges of about 211 fluorescence bursting spikes from the trajectoriesshown in Figure 2. Here, the signal risingedge corresponds to the fluorescent product generated at a single-moleculereactant-to-product turnover, and the signal falling edge correspondsto the product releasing from the active site into the solution anddiffusing away from the confocal volume. Figure 6 shows the distributions of the lifetimes and anisotropies of theproducts from the rising edges and the falling edges of each fluorescencespike. Comparing the lifetime distributions from the rising and fallingedges of the fluorescence spikes (Figure 6 A1and Figure 6 B1), we notice that the mean lifetimeof the falling part is about 0.38 ns shorter than that of the risingpart, while the anisotropy distributions are almost the same. On thebasis of the influence of the quenching effect and local dielectricconstant on the fluorescence lifetime of the nascent product as discussedabove, the most possible explanation for the shorter lifetime of thesingle resorufin molecules at the falling edges is that the productmolecules are buried in tight states compared to the rising edge.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University , Bowling Green, Ohio 43403, United States.

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.

Show MeSH
Related in: MedlinePlus