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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.

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Correlationplots of the lifetime, anisotropy, and intensity onthe single HRP-catalyzed amplex red fluorogenic assay. (A1) Correlationof fluorescence intensity and anisotropy and (B1) correlation of lifetimeand anisotropy. For A1 and B1, the color (from cold color to warmcolor, i.e., from blue to red) of the data points represents the timesequence from the start to the end of the fluorogenic enzymatic turnoverevent. (A2) The correlation plots of fluorescence intensity and anisotropywith identified rotational correlation times. (B2) Correlation oflifetime and anisotropy with identified rotational correlation times.
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fig5: Correlationplots of the lifetime, anisotropy, and intensity onthe single HRP-catalyzed amplex red fluorogenic assay. (A1) Correlationof fluorescence intensity and anisotropy and (B1) correlation of lifetimeand anisotropy. For A1 and B1, the color (from cold color to warmcolor, i.e., from blue to red) of the data points represents the timesequence from the start to the end of the fluorogenic enzymatic turnoverevent. (A2) The correlation plots of fluorescence intensity and anisotropywith identified rotational correlation times. (B2) Correlation oflifetime and anisotropy with identified rotational correlation times.

Mentions: Performing correlation analysisof the lifetime, anisotropy, andintensity on the single-molecule photon-by-photon trajectories allowsus to characterize the complexity and heterogeneity of enzymatic conformationaldynamics and mechanism.37,65,66 Figures 5 A1 and 5 B1 show the correlated distributions of intensity vs anisotropyand fluorescence lifetime vs anisotropy deduced from the trajectoriesshown in Figure 3 and Figure 4, respectively. Figures 5 A2 and 5 B2 show the correlated counter plots of Figures 5 A1 and 5 B1. There are distinctlythree domains in the intensity–anisotropy 2D plot (Figure 5A2), while in the lifetime–anisotropy 2Dplot (Figure 5 B2), there are two domains 3′and 3″ separated at the high anisotropy value, except the samedomains 1 and 2 as in Figure 5 A2. These domainsreflect the different local environments of the nascent fluorescentenzymatic reaction product, resorufin, at the enzymatic active site.To assign the origin of each domain in the correlation distribution2D plots, the rotational correlation times (τr) areneeded as shown in eq 6.


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

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

Correlationplots of the lifetime, anisotropy, and intensity onthe single HRP-catalyzed amplex red fluorogenic assay. (A1) Correlationof fluorescence intensity and anisotropy and (B1) correlation of lifetimeand anisotropy. For A1 and B1, the color (from cold color to warmcolor, i.e., from blue to red) of the data points represents the timesequence from the start to the end of the fluorogenic enzymatic turnoverevent. (A2) The correlation plots of fluorescence intensity and anisotropywith identified rotational correlation times. (B2) Correlation oflifetime and anisotropy with identified rotational correlation times.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Correlationplots of the lifetime, anisotropy, and intensity onthe single HRP-catalyzed amplex red fluorogenic assay. (A1) Correlationof fluorescence intensity and anisotropy and (B1) correlation of lifetimeand anisotropy. For A1 and B1, the color (from cold color to warmcolor, i.e., from blue to red) of the data points represents the timesequence from the start to the end of the fluorogenic enzymatic turnoverevent. (A2) The correlation plots of fluorescence intensity and anisotropywith identified rotational correlation times. (B2) Correlation oflifetime and anisotropy with identified rotational correlation times.
Mentions: Performing correlation analysisof the lifetime, anisotropy, andintensity on the single-molecule photon-by-photon trajectories allowsus to characterize the complexity and heterogeneity of enzymatic conformationaldynamics and mechanism.37,65,66 Figures 5 A1 and 5 B1 show the correlated distributions of intensity vs anisotropyand fluorescence lifetime vs anisotropy deduced from the trajectoriesshown in Figure 3 and Figure 4, respectively. Figures 5 A2 and 5 B2 show the correlated counter plots of Figures 5 A1 and 5 B1. There are distinctlythree domains in the intensity–anisotropy 2D plot (Figure 5A2), while in the lifetime–anisotropy 2Dplot (Figure 5 B2), there are two domains 3′and 3″ separated at the high anisotropy value, except the samedomains 1 and 2 as in Figure 5 A2. These domainsreflect the different local environments of the nascent fluorescentenzymatic reaction product, resorufin, at the enzymatic active site.To assign the origin of each domain in the correlation distribution2D plots, the rotational correlation times (τr) areneeded as shown in eq 6.

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