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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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Single-molecule fluorescence anisotropyfluctuation and distributionof single HRP-catalyzed oxidation of Amplex Red fluorogenic assay.(A) Single-molecule fluorescence anisotropy fluctuation. Intensitytrajectory from the perpendicular polarization component relativeto the polarization of excitation (green) and the simultaneously recordedintensity trajectory from the parallel polarization component relativeto the polarization of excitation (red); the calculated anisotropytrajectory (blue) using eq 2 from the pair ofpolarization components (red and green). (B) Anisotropy distributionfrom the trajectory (blue) in A. The anisotropy distribution fromthe background (black) with mean of −0.020 and standard deviationof 0.054. It is evident that the signal anisotropy distribution (blue)is identifiable, beyond the background distribution.
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fig3: Single-molecule fluorescence anisotropyfluctuation and distributionof single HRP-catalyzed oxidation of Amplex Red fluorogenic assay.(A) Single-molecule fluorescence anisotropy fluctuation. Intensitytrajectory from the perpendicular polarization component relativeto the polarization of excitation (green) and the simultaneously recordedintensity trajectory from the parallel polarization component relativeto the polarization of excitation (red); the calculated anisotropytrajectory (blue) using eq 2 from the pair ofpolarization components (red and green). (B) Anisotropy distributionfrom the trajectory (blue) in A. The anisotropy distribution fromthe background (black) with mean of −0.020 and standard deviationof 0.054. It is evident that the signal anisotropy distribution (blue)is identifiable, beyond the background distribution.

Mentions: Anisotropy provides a critical analysis of therotational dynamicsof a fluorescent molecule; furthermore, specific physical parametersof the local environment influencing the probe molecule rotationalmotions can also be either qualitatively or semiquantitatively characterized,such as the free space, force field, electric field, hydrophobicity,hydrodynamics, solvation effect, and hydrodynamic volume changes.Figure 3A shows the anisotropy fluctuationtrajectory and the corresponding polarization intensity trajectoryof a portion of typical single-molecule photon stamping trajectories(Figure 2). Here the intensity is calculatedfrom 300 photons divided by the time intervals for accumulating theconsecutive 300 photons to control the shot noise at the same level.The anisotropy shows as constant around zero between the fluorescencephoton bursting. During the bursting, the anisotropy shows a dramaticfluctuation. Figure 3B shows the correspondinganisotropy distribution with two distinct peaks. The anisotropy distributionaround the peak near zero has a narrow Gaussian-like shape and isdominated by the background, as compared to the control backgroundwith mean at −0.02 and with standard deviation as 0.054, asshown in the black curve. The anisotropy distribution at higher anisotropybeyond the background is asymmetric and elongated toward higher values.This broad distribution of the high anisotropy spans from 0.1 and0.36, which is larger than the standard deviation of the background.To identify the specific interactions of the enzyme–substrateand enzyme–product at the enzymatic active site, it is reasonableto assume that the substrate amplex red and the nascent fluorescentproduct bind to the active site in a similar configuration as a typicalaromatic substrate does, such as a reported binding structure of aHRP–acetate complex.23,25 Aromatic substratesform stable, reversible 1:1 complexes with HRP through both hydrogen-bondedand hydrophobic interactions at the distal side of the heme plane.The amino acid residues Arg38 and His42 play the roles in bindingand stabilization of aromatic substrates (see Supporting Information). At the enzymatic active site of theHRP–resorufin complex, the rigid hydrogen bonding of resorufinto the HRP enzyme ensures that the HRP product active site rotatesrelatively confined, which in turn makes the measured fluorescenceanisotropy trajectories likely to yield information about the rotationaland conformational dynamics of not only the fluorescent product butalso the HRP protein, particularly of the solvent exposed heme prostheticgroup at the active site, where the oxidations of aromatic substrateshappen. The broad distribution of the anisotropy in Figure 3B also indicates that likely multiple conformationalstates are involved in active-site interaction with the product moleculesduring the product releasing, which is consistent with the bindingsite being a relatively flexible structural region.


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

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

Single-molecule fluorescence anisotropyfluctuation and distributionof single HRP-catalyzed oxidation of Amplex Red fluorogenic assay.(A) Single-molecule fluorescence anisotropy fluctuation. Intensitytrajectory from the perpendicular polarization component relativeto the polarization of excitation (green) and the simultaneously recordedintensity trajectory from the parallel polarization component relativeto the polarization of excitation (red); the calculated anisotropytrajectory (blue) using eq 2 from the pair ofpolarization components (red and green). (B) Anisotropy distributionfrom the trajectory (blue) in A. The anisotropy distribution fromthe background (black) with mean of −0.020 and standard deviationof 0.054. It is evident that the signal anisotropy distribution (blue)is identifiable, beyond the background distribution.
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Related In: Results  -  Collection

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

fig3: Single-molecule fluorescence anisotropyfluctuation and distributionof single HRP-catalyzed oxidation of Amplex Red fluorogenic assay.(A) Single-molecule fluorescence anisotropy fluctuation. Intensitytrajectory from the perpendicular polarization component relativeto the polarization of excitation (green) and the simultaneously recordedintensity trajectory from the parallel polarization component relativeto the polarization of excitation (red); the calculated anisotropytrajectory (blue) using eq 2 from the pair ofpolarization components (red and green). (B) Anisotropy distributionfrom the trajectory (blue) in A. The anisotropy distribution fromthe background (black) with mean of −0.020 and standard deviationof 0.054. It is evident that the signal anisotropy distribution (blue)is identifiable, beyond the background distribution.
Mentions: Anisotropy provides a critical analysis of therotational dynamicsof a fluorescent molecule; furthermore, specific physical parametersof the local environment influencing the probe molecule rotationalmotions can also be either qualitatively or semiquantitatively characterized,such as the free space, force field, electric field, hydrophobicity,hydrodynamics, solvation effect, and hydrodynamic volume changes.Figure 3A shows the anisotropy fluctuationtrajectory and the corresponding polarization intensity trajectoryof a portion of typical single-molecule photon stamping trajectories(Figure 2). Here the intensity is calculatedfrom 300 photons divided by the time intervals for accumulating theconsecutive 300 photons to control the shot noise at the same level.The anisotropy shows as constant around zero between the fluorescencephoton bursting. During the bursting, the anisotropy shows a dramaticfluctuation. Figure 3B shows the correspondinganisotropy distribution with two distinct peaks. The anisotropy distributionaround the peak near zero has a narrow Gaussian-like shape and isdominated by the background, as compared to the control backgroundwith mean at −0.02 and with standard deviation as 0.054, asshown in the black curve. The anisotropy distribution at higher anisotropybeyond the background is asymmetric and elongated toward higher values.This broad distribution of the high anisotropy spans from 0.1 and0.36, which is larger than the standard deviation of the background.To identify the specific interactions of the enzyme–substrateand enzyme–product at the enzymatic active site, it is reasonableto assume that the substrate amplex red and the nascent fluorescentproduct bind to the active site in a similar configuration as a typicalaromatic substrate does, such as a reported binding structure of aHRP–acetate complex.23,25 Aromatic substratesform stable, reversible 1:1 complexes with HRP through both hydrogen-bondedand hydrophobic interactions at the distal side of the heme plane.The amino acid residues Arg38 and His42 play the roles in bindingand stabilization of aromatic substrates (see Supporting Information). At the enzymatic active site of theHRP–resorufin complex, the rigid hydrogen bonding of resorufinto the HRP enzyme ensures that the HRP product active site rotatesrelatively confined, which in turn makes the measured fluorescenceanisotropy trajectories likely to yield information about the rotationaland conformational dynamics of not only the fluorescent product butalso the HRP protein, particularly of the solvent exposed heme prostheticgroup at the active site, where the oxidations of aromatic substrateshappen. The broad distribution of the anisotropy in Figure 3B also indicates that likely multiple conformationalstates are involved in active-site interaction with the product moleculesduring the product releasing, which is consistent with the bindingsite being a relatively flexible structural region.

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