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Structural basis for certain naturally occurring bioflavonoids to function as reducing co-substrates of cyclooxygenase I and II.

Wang P, Bai HW, Zhu BT - PLoS ONE (2010)

Bottom Line: The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity.Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

ABSTRACT

Background: Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds.

Methodology/principal findings: A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.

Conclusions/significance: These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation.

Show MeSH
Schematic depiction of the catalysis and inactivation mechanism of COX enzymes and their interaction with bioflavonoids.PPIX is for protoporphorin IX. Quercetin structure is shown as a representative bioactive bioflavonoid. Events in the peroxidase cycle are labeled with numbers to denote the sequence of occurrence.
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pone-0012316-g006: Schematic depiction of the catalysis and inactivation mechanism of COX enzymes and their interaction with bioflavonoids.PPIX is for protoporphorin IX. Quercetin structure is shown as a representative bioactive bioflavonoid. Events in the peroxidase cycle are labeled with numbers to denote the sequence of occurrence.

Mentions: There were several earlier studies that investigated a number of reducing co-substrates of COXs [22]–[26]. The mechanism is generally thought to be due to the reduction of the oxidized intermediates by the co-substrates [23], [25]. As depicted in Figure 6, the reducing potential of bioflavonoids, like other reducing co-substrates, will help maintain the peroxidase cycle and thereby slow down the suicidal inactivation of the COX enzymes by donating one electron each to Compound I and II to restore the reducing activity of hematin, which is needed for the peroxidase to convert PGG2 to PGH2. The oxidized quinone form of bioflavonoids is expected to have a lower binding affinity for the peroxidase site because they will lose two hydrogen bond donors (hydroxyl groups), and some of the hydrogen bonds cannot be formed between the quinine form and the peroxidase site. Accordingly, the following catalytic sequence is proposed (depicted in Figure 6): It is assumed that PGG2 has a high binding affinity for the peroxidase site of the enzyme and will tightly bind to this site. Immediately following the catalytic conversion of PGG2 to its product, the product will dissociate from the enzyme (due to a reduced binding affinity). After that, the peroxidase site will become catalytically inactive (with an oxidized hematin), and it will be bound by a bioflavonoid molecule (in its reduced form) for the reduction of hematin to its initial state. During the process, the bioflavonoid is oxidized initially to semiquione (as an intermediate) and then to quinone. The bioflavonoid quinone will then be released from the activated peroxidase site because the oxidized molecule will have a reduced binding affinity for the peroxidase active site. In this model, it is apparent that there is a potential competition between the substrate (PGG2) and the co-substrate (bioflavonoid) at the peroxidase catalytic site. When the bioflavonoid concentration becomes too high, it will increase the fraction of the active peroxidase site that is still occupied by the reducing co-substrate, and when this occurs, it would inhibit the binding of PGG2 to the peroxidase site and thus would reduce the catalytic activity of the enzyme for the formation of further products. This mechanistic explanation is in agreement with the data shown in Figure 5C and 5F as well as a number of earlier studies showing a concentration-dependent biphasic modulation of the COX activity by co-substrates, namely, the presence of a co-substrate at low concentrations stimulated the COX activity, whereas its presence at higher concentrations inhibited the COX activity [22]–[28]. Notably, some of the earlier studies suggested that the inhibition by high concentrations of the co-substrate was due to the fast reduction of Intermediate II and the loss of cyclooxygenase activity [27]. This explanation appears to disagree with the observation (in Figure 5C and 5F) made in this study which showed that myricetin at high concentrations (500 and 1000 µM) inhibited the peroxidase activity of aspirin-pretreated COX II (using PGG2 as substrate) to a similar degree as they inhibited the catalytic activity of untreated COX II enzyme for its metabolism of AA as substrate.


Structural basis for certain naturally occurring bioflavonoids to function as reducing co-substrates of cyclooxygenase I and II.

Wang P, Bai HW, Zhu BT - PLoS ONE (2010)

Schematic depiction of the catalysis and inactivation mechanism of COX enzymes and their interaction with bioflavonoids.PPIX is for protoporphorin IX. Quercetin structure is shown as a representative bioactive bioflavonoid. Events in the peroxidase cycle are labeled with numbers to denote the sequence of occurrence.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2925883&req=5

pone-0012316-g006: Schematic depiction of the catalysis and inactivation mechanism of COX enzymes and their interaction with bioflavonoids.PPIX is for protoporphorin IX. Quercetin structure is shown as a representative bioactive bioflavonoid. Events in the peroxidase cycle are labeled with numbers to denote the sequence of occurrence.
Mentions: There were several earlier studies that investigated a number of reducing co-substrates of COXs [22]–[26]. The mechanism is generally thought to be due to the reduction of the oxidized intermediates by the co-substrates [23], [25]. As depicted in Figure 6, the reducing potential of bioflavonoids, like other reducing co-substrates, will help maintain the peroxidase cycle and thereby slow down the suicidal inactivation of the COX enzymes by donating one electron each to Compound I and II to restore the reducing activity of hematin, which is needed for the peroxidase to convert PGG2 to PGH2. The oxidized quinone form of bioflavonoids is expected to have a lower binding affinity for the peroxidase site because they will lose two hydrogen bond donors (hydroxyl groups), and some of the hydrogen bonds cannot be formed between the quinine form and the peroxidase site. Accordingly, the following catalytic sequence is proposed (depicted in Figure 6): It is assumed that PGG2 has a high binding affinity for the peroxidase site of the enzyme and will tightly bind to this site. Immediately following the catalytic conversion of PGG2 to its product, the product will dissociate from the enzyme (due to a reduced binding affinity). After that, the peroxidase site will become catalytically inactive (with an oxidized hematin), and it will be bound by a bioflavonoid molecule (in its reduced form) for the reduction of hematin to its initial state. During the process, the bioflavonoid is oxidized initially to semiquione (as an intermediate) and then to quinone. The bioflavonoid quinone will then be released from the activated peroxidase site because the oxidized molecule will have a reduced binding affinity for the peroxidase active site. In this model, it is apparent that there is a potential competition between the substrate (PGG2) and the co-substrate (bioflavonoid) at the peroxidase catalytic site. When the bioflavonoid concentration becomes too high, it will increase the fraction of the active peroxidase site that is still occupied by the reducing co-substrate, and when this occurs, it would inhibit the binding of PGG2 to the peroxidase site and thus would reduce the catalytic activity of the enzyme for the formation of further products. This mechanistic explanation is in agreement with the data shown in Figure 5C and 5F as well as a number of earlier studies showing a concentration-dependent biphasic modulation of the COX activity by co-substrates, namely, the presence of a co-substrate at low concentrations stimulated the COX activity, whereas its presence at higher concentrations inhibited the COX activity [22]–[28]. Notably, some of the earlier studies suggested that the inhibition by high concentrations of the co-substrate was due to the fast reduction of Intermediate II and the loss of cyclooxygenase activity [27]. This explanation appears to disagree with the observation (in Figure 5C and 5F) made in this study which showed that myricetin at high concentrations (500 and 1000 µM) inhibited the peroxidase activity of aspirin-pretreated COX II (using PGG2 as substrate) to a similar degree as they inhibited the catalytic activity of untreated COX II enzyme for its metabolism of AA as substrate.

Bottom Line: The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity.Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

ABSTRACT

Background: Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds.

Methodology/principal findings: A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.

Conclusions/significance: These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation.

Show MeSH