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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 fluorescencelifetime fluctuation and distributionof single HRP-catalyzed amplex red fluorogenic assay. (A) Single-moleculefluorescence lifetime fluctuation trajectory. Intensity trajectoryfrom the parallel polarization component relative to the polarizationof excitation (red) and the simultaneously recorded intensity trajectoryfrom the perpendicular polarization component relative to the polarizationof excitation (green). Lifetime fluctuation trajectory (blue) calculatedusing eq 5 from the pair of polarization componenttrajectories (red and green). (B) Single-molecule enzymatic reactionproduct fluorescence lifetime distribution and the lifetime backgrounddistribution (black) from the trajectory (blue) in A. The lifetimebackground distribution is deduced from the lifetime trajectory from678 to 681.5 s in (A), and the background distribution gives the meanof 3.01 ns and standard deviation of 0.60 ns. (C1), (C2), and (C3)Fluorescence decays at point 1 at 678.01 s, point 2 at 682.29 s, andpoint 3 at 684.75 s, respectively. The parallel polarization componentdecays (red), the perpendicular polarization component decays (green),and the fluorescence decays with fit (blue). The lifetimes at point1, point 2, and point 3 are 2.9 ± 0.2 ns, 2.7 ± 0.1 ns,and 4.1 ± 0.1 ns, respectively.
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fig4: Single-molecule fluorescencelifetime fluctuation and distributionof single HRP-catalyzed amplex red fluorogenic assay. (A) Single-moleculefluorescence lifetime fluctuation trajectory. Intensity trajectoryfrom the parallel polarization component relative to the polarizationof excitation (red) and the simultaneously recorded intensity trajectoryfrom the perpendicular polarization component relative to the polarizationof excitation (green). Lifetime fluctuation trajectory (blue) calculatedusing eq 5 from the pair of polarization componenttrajectories (red and green). (B) Single-molecule enzymatic reactionproduct fluorescence lifetime distribution and the lifetime backgrounddistribution (black) from the trajectory (blue) in A. The lifetimebackground distribution is deduced from the lifetime trajectory from678 to 681.5 s in (A), and the background distribution gives the meanof 3.01 ns and standard deviation of 0.60 ns. (C1), (C2), and (C3)Fluorescence decays at point 1 at 678.01 s, point 2 at 682.29 s, andpoint 3 at 684.75 s, respectively. The parallel polarization componentdecays (red), the perpendicular polarization component decays (green),and the fluorescence decays with fit (blue). The lifetimes at point1, point 2, and point 3 are 2.9 ± 0.2 ns, 2.7 ± 0.1 ns,and 4.1 ± 0.1 ns, respectively.

Mentions: Fluorescence lifetime can be determined independently frommolecularrotation through the linear combination of eqs 3 and 4. Figure 4A showsthe lifetime fluctuation trajectory and the corresponding polarizationintensity trajectories from a portion of the photon stamping trajectoriesin Figure 2. Each data point in the lifetimetrajectory is calculated from the consecutive 300 photons of the twotime-resolved photon stamping polarization channels. Figure 4B shows the lifetime distribution of the fluctuationlifetime trajectory in Figure 4A. There areno evident multipeaks in the lifetime distribution as that in theanisotropy distribution. The lifetime background distribution (Figure 4B) shows the mean at 3.0 ± 0.6 ns, which isconsistent with the lifetime of free resorufin molecules in pH 7.4buffer. Compared to the lifetime of the background, the average lifetimeof the nascent enzymatic reaction product, resorufin, is much longerthan that of free resorufin molecules (Figure 4B). Since the lifetime of the resorufin is sensitive to the pH ofthe solution,54 the longer average lifetimeof the confined resorufin is likely influenced by the local basicityof the active site. This is consistent with the fact that the hydrogenbond from amide oxygen of amino acid residue Asn70 to imidazole NHof amino acid residue His42 contributes to the basicity of the localenvironment.23 Since the florescence lifetimeis sensitive to the fluctuation of the local environment and the movementsof macromolecules,52,55−59 the lifetime fluctuations of the confined resorufinmost likely reflect the fluctuations of the active-site conformationof the HRP enzyme as well as the enzyme–product molecular interactions.Figure 4 C1 shows the typical fluorescencedecay behavior of the free resorufin molecules from point 1 at 678.01s in Figure 4A. The fluorescence intensityof parallel polarization shows similar decay as that of the perpendicularpolarization component, which is consistent with the rotation of thefree resorufin molecules being faster than the fluorescence lifetime.60,61 Figures 4 C2 and 4 C3 have shown the fluorescence decay behaviors during the burstingfrom point 2 at 682.29 s and point 3 at 684.75 s, respectively, inthe trajectory 4A. At point 2, the lifetime is shorter than that ofthe free resorufin molecules in buffer solution, and the shorter lifetimeis likely due to the quenching effect from the heme group at the enzymaticactive site, since there exists the energy transfer between the enzymaticproduct, resorufin, and the heme group.56,57,62,63 At point 3, the fluorescencedecay shows a longer lifetime than that in point 2. The significantincrease of the lifetime suggests a significant change of the enzyme–productmolecular interaction at the active site change, presumably makingthe nascent product molecule more efficiently prevented by the Phe41residue to access the heme iron,25 whichdecreases the energy transfer between the product and the heme group.At the enzymatic active site and at the particular time of point 3the product most likely exists as the resorufin anion (R–) state since it is dramatically different in lifetime compared tothat of the protonated form of resorufin (RH).54,64


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

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

Single-molecule fluorescencelifetime fluctuation and distributionof single HRP-catalyzed amplex red fluorogenic assay. (A) Single-moleculefluorescence lifetime fluctuation trajectory. Intensity trajectoryfrom the parallel polarization component relative to the polarizationof excitation (red) and the simultaneously recorded intensity trajectoryfrom the perpendicular polarization component relative to the polarizationof excitation (green). Lifetime fluctuation trajectory (blue) calculatedusing eq 5 from the pair of polarization componenttrajectories (red and green). (B) Single-molecule enzymatic reactionproduct fluorescence lifetime distribution and the lifetime backgrounddistribution (black) from the trajectory (blue) in A. The lifetimebackground distribution is deduced from the lifetime trajectory from678 to 681.5 s in (A), and the background distribution gives the meanof 3.01 ns and standard deviation of 0.60 ns. (C1), (C2), and (C3)Fluorescence decays at point 1 at 678.01 s, point 2 at 682.29 s, andpoint 3 at 684.75 s, respectively. The parallel polarization componentdecays (red), the perpendicular polarization component decays (green),and the fluorescence decays with fit (blue). The lifetimes at point1, point 2, and point 3 are 2.9 ± 0.2 ns, 2.7 ± 0.1 ns,and 4.1 ± 0.1 ns, respectively.
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fig4: Single-molecule fluorescencelifetime fluctuation and distributionof single HRP-catalyzed amplex red fluorogenic assay. (A) Single-moleculefluorescence lifetime fluctuation trajectory. Intensity trajectoryfrom the parallel polarization component relative to the polarizationof excitation (red) and the simultaneously recorded intensity trajectoryfrom the perpendicular polarization component relative to the polarizationof excitation (green). Lifetime fluctuation trajectory (blue) calculatedusing eq 5 from the pair of polarization componenttrajectories (red and green). (B) Single-molecule enzymatic reactionproduct fluorescence lifetime distribution and the lifetime backgrounddistribution (black) from the trajectory (blue) in A. The lifetimebackground distribution is deduced from the lifetime trajectory from678 to 681.5 s in (A), and the background distribution gives the meanof 3.01 ns and standard deviation of 0.60 ns. (C1), (C2), and (C3)Fluorescence decays at point 1 at 678.01 s, point 2 at 682.29 s, andpoint 3 at 684.75 s, respectively. The parallel polarization componentdecays (red), the perpendicular polarization component decays (green),and the fluorescence decays with fit (blue). The lifetimes at point1, point 2, and point 3 are 2.9 ± 0.2 ns, 2.7 ± 0.1 ns,and 4.1 ± 0.1 ns, respectively.
Mentions: Fluorescence lifetime can be determined independently frommolecularrotation through the linear combination of eqs 3 and 4. Figure 4A showsthe lifetime fluctuation trajectory and the corresponding polarizationintensity trajectories from a portion of the photon stamping trajectoriesin Figure 2. Each data point in the lifetimetrajectory is calculated from the consecutive 300 photons of the twotime-resolved photon stamping polarization channels. Figure 4B shows the lifetime distribution of the fluctuationlifetime trajectory in Figure 4A. There areno evident multipeaks in the lifetime distribution as that in theanisotropy distribution. The lifetime background distribution (Figure 4B) shows the mean at 3.0 ± 0.6 ns, which isconsistent with the lifetime of free resorufin molecules in pH 7.4buffer. Compared to the lifetime of the background, the average lifetimeof the nascent enzymatic reaction product, resorufin, is much longerthan that of free resorufin molecules (Figure 4B). Since the lifetime of the resorufin is sensitive to the pH ofthe solution,54 the longer average lifetimeof the confined resorufin is likely influenced by the local basicityof the active site. This is consistent with the fact that the hydrogenbond from amide oxygen of amino acid residue Asn70 to imidazole NHof amino acid residue His42 contributes to the basicity of the localenvironment.23 Since the florescence lifetimeis sensitive to the fluctuation of the local environment and the movementsof macromolecules,52,55−59 the lifetime fluctuations of the confined resorufinmost likely reflect the fluctuations of the active-site conformationof the HRP enzyme as well as the enzyme–product molecular interactions.Figure 4 C1 shows the typical fluorescencedecay behavior of the free resorufin molecules from point 1 at 678.01s in Figure 4A. The fluorescence intensityof parallel polarization shows similar decay as that of the perpendicularpolarization component, which is consistent with the rotation of thefree resorufin molecules being faster than the fluorescence lifetime.60,61 Figures 4 C2 and 4 C3 have shown the fluorescence decay behaviors during the burstingfrom point 2 at 682.29 s and point 3 at 684.75 s, respectively, inthe trajectory 4A. At point 2, the lifetime is shorter than that ofthe free resorufin molecules in buffer solution, and the shorter lifetimeis likely due to the quenching effect from the heme group at the enzymaticactive site, since there exists the energy transfer between the enzymaticproduct, resorufin, and the heme group.56,57,62,63 At point 3, the fluorescencedecay shows a longer lifetime than that in point 2. The significantincrease of the lifetime suggests a significant change of the enzyme–productmolecular interaction at the active site change, presumably makingthe nascent product molecule more efficiently prevented by the Phe41residue to access the heme iron,25 whichdecreases the energy transfer between the product and the heme group.At the enzymatic active site and at the particular time of point 3the product most likely exists as the resorufin anion (R–) state since it is dramatically different in lifetime compared tothat of the protonated form of resorufin (RH).54,64

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