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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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Proposed dynamics of single HRP-catalyzedoxidation of amplex redreaction. (A) The breathing motions of the enzymatic reaction activesite. (B) The schematic of the two pathways of product release. Ourexperimental observation suggests that about 20% product releasingis involved in the spilling pathways.
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fig8: Proposed dynamics of single HRP-catalyzedoxidation of amplex redreaction. (A) The breathing motions of the enzymatic reaction activesite. (B) The schematic of the two pathways of product release. Ourexperimental observation suggests that about 20% product releasingis involved in the spilling pathways.

Mentions: It is well-known that enzymatic reactions involve multiplekineticsteps, and each step involves complex molecular interactions, inhomogeneousconformational changes, and fluctuations of confined local environment.6−9 Both the overall dynamics and mechanism of the enzymatic reactionhave been well investigated in ensemble-averaged experiments aboutthe HRP enzyme, although there are different fluorogenic enzymaticreaction mechanisms proposed in the recent literature (see Supporting Information).23−26,30,85−89 Here, the catalytic networks of the HRP-catalyzedamplex red have been further characterized by our single-moleculefluorogenic assay. Rotational correlation times of the product duringthe releasing process have shown the enzyme undergoing breathing-typeconformational motions fluctuating between loose states and the tightstates. On the basis of the crystal structure of HRP, these breathing-typeconfigurational motions may come from the active-site segment motionas shown in Figure 8A. As the enzyme changesto the loose states, the water-soluble substrate is facilitated totransport into the active site. Conformation fluctuations betweenthe loose states and tight states occur during the products releasing.Nevertheless, we propose a new mechanism of the HRP enzymatic reaction(Figure 8B): There are multiple conformationsinvolved in the enzymatic reaction enzyme–substrate and enzyme–productinteractions as well as the product releasing from the active site,and the conformational fluctuations occur in consecutive and parallelpathways in the mechanism with both the active-site open releasingproduct pathway and the active-site close spilling product pathway.7 Overall, the active-site close spilling productpathway reported in this work is the most significant new knowledgefor the HRP enzymatic reaction dynamics and mechanism. We anticipatedthat the “spilling” product releasing pathway widelyexisted in other enzymatic reaction product releasings, especiallyfor the enzyme active-site associated with large conformational motionsin enzymatic reaction turnovers.


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

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

Proposed dynamics of single HRP-catalyzedoxidation of amplex redreaction. (A) The breathing motions of the enzymatic reaction activesite. (B) The schematic of the two pathways of product release. Ourexperimental observation suggests that about 20% product releasingis involved in the spilling pathways.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Proposed dynamics of single HRP-catalyzedoxidation of amplex redreaction. (A) The breathing motions of the enzymatic reaction activesite. (B) The schematic of the two pathways of product release. Ourexperimental observation suggests that about 20% product releasingis involved in the spilling pathways.
Mentions: It is well-known that enzymatic reactions involve multiplekineticsteps, and each step involves complex molecular interactions, inhomogeneousconformational changes, and fluctuations of confined local environment.6−9 Both the overall dynamics and mechanism of the enzymatic reactionhave been well investigated in ensemble-averaged experiments aboutthe HRP enzyme, although there are different fluorogenic enzymaticreaction mechanisms proposed in the recent literature (see Supporting Information).23−26,30,85−89 Here, the catalytic networks of the HRP-catalyzedamplex red have been further characterized by our single-moleculefluorogenic assay. Rotational correlation times of the product duringthe releasing process have shown the enzyme undergoing breathing-typeconformational motions fluctuating between loose states and the tightstates. On the basis of the crystal structure of HRP, these breathing-typeconfigurational motions may come from the active-site segment motionas shown in Figure 8A. As the enzyme changesto the loose states, the water-soluble substrate is facilitated totransport into the active site. Conformation fluctuations betweenthe loose states and tight states occur during the products releasing.Nevertheless, we propose a new mechanism of the HRP enzymatic reaction(Figure 8B): There are multiple conformationsinvolved in the enzymatic reaction enzyme–substrate and enzyme–productinteractions as well as the product releasing from the active site,and the conformational fluctuations occur in consecutive and parallelpathways in the mechanism with both the active-site open releasingproduct pathway and the active-site close spilling product pathway.7 Overall, the active-site close spilling productpathway reported in this work is the most significant new knowledgefor the HRP enzymatic reaction dynamics and mechanism. We anticipatedthat the “spilling” product releasing pathway widelyexisted in other enzymatic reaction product releasings, especiallyfor the enzyme active-site associated with large conformational motionsin enzymatic reaction turnovers.

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