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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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Distributionof rotation correlation time of the product at thesignal falling edges that correspond to the moments of releasing theproduct molecules from the enzyme into the solution.
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fig7: Distributionof rotation correlation time of the product at thesignal falling edges that correspond to the moments of releasing theproduct molecules from the enzyme into the solution.

Mentions: Rotation correlation time of the enzymatic reactionproduct, resorufin,at the falling edges of the turnover fluorogenic signals is a sensitiveparameter to reveal the enzyme–product interactions right atthe product releasing events. Figure 7 showsthe distribution of the rotation correlation time of the resorufinat the falling edges and the distribution peaks at 2 ns that are closeto the rotation correlation time of the loose enzyme–productstates in region 2 of Figure 5B2, and theseproduct events are most likely related to the enzyme active site openingup to release the fluorescent product into the solution. Nevertheless,about 20% of the product releasing events are associated with therotation correlation time larger than 9 ns, which suggests that theproduct releasing events occurred at a strong enzyme–productinteraction so that the fluorescent product rotational motions areat the protein motion time scale. Furthermore, the fluorescence lifetimeof the product releasing events is also associated with shorter fluorescencelifetime, as we have discussed above, which also suggests a tightenzyme–substrate interaction at the events of product releasing.The correlated shorter lifetime and longer rotation correlation timeof the products at the falling edges support our attribution thatthere is a significant pathway in which the product molecules arespilled out from the enzymatic active site, driven by a squeezingeffect by the breath-type motions between loose and tight active-siteconformation states; i.e., there is a significant portion of productmolecules that are released from a tight active-site conformationalstate rather than from an open active-site conformational state. Thismost remarkable phenomenon provides a new insight into the enzymaticreaction dynamics and mechanism—specifically on the productreleasing mechanism and dynamics.


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

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

Distributionof rotation correlation time of the product at thesignal falling edges that correspond to the moments of releasing theproduct molecules from the enzyme into the solution.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Distributionof rotation correlation time of the product at thesignal falling edges that correspond to the moments of releasing theproduct molecules from the enzyme into the solution.
Mentions: Rotation correlation time of the enzymatic reactionproduct, resorufin,at the falling edges of the turnover fluorogenic signals is a sensitiveparameter to reveal the enzyme–product interactions right atthe product releasing events. Figure 7 showsthe distribution of the rotation correlation time of the resorufinat the falling edges and the distribution peaks at 2 ns that are closeto the rotation correlation time of the loose enzyme–productstates in region 2 of Figure 5B2, and theseproduct events are most likely related to the enzyme active site openingup to release the fluorescent product into the solution. Nevertheless,about 20% of the product releasing events are associated with therotation correlation time larger than 9 ns, which suggests that theproduct releasing events occurred at a strong enzyme–productinteraction so that the fluorescent product rotational motions areat the protein motion time scale. Furthermore, the fluorescence lifetimeof the product releasing events is also associated with shorter fluorescencelifetime, as we have discussed above, which also suggests a tightenzyme–substrate interaction at the events of product releasing.The correlated shorter lifetime and longer rotation correlation timeof the products at the falling edges support our attribution thatthere is a significant pathway in which the product molecules arespilled out from the enzymatic active site, driven by a squeezingeffect by the breath-type motions between loose and tight active-siteconformation states; i.e., there is a significant portion of productmolecules that are released from a tight active-site conformationalstate rather than from an open active-site conformational state. Thismost remarkable phenomenon provides a new insight into the enzymaticreaction dynamics and mechanism—specifically on the productreleasing mechanism and dynamics.

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