Modeling of biopterin-dependent pathways of eNOS for nitric oxide and superoxide production

Abstract

Endothelial dysfunction is associated with increase in oxidative stress and low NO bioavailability. The endothelial NO synthase (eNOS) uncoupling is considered an important factor in endothelial cell oxidative stress. Under increased oxidative stress, the eNOS cofactor tetrahydrobiopterin (BH(4)) is oxidized to dihydrobiopterin, which competes with BH(4) for binding to eNOS, resulting in eNOS uncoupling and reduction in NO production. The importance of the ratio of BH(4) to oxidized biopterins versus absolute levels of total biopterin in determining the extent of eNOS uncoupling remains to be determined. We have developed a computational model to simulate the kinetics of the biochemical pathways of eNOS for both NO and O(2)(•-) production to understand the roles of BH(4) availability and total biopterin (TBP) concentration in eNOS uncoupling. The downstream reactions of NO, O(2)(•-), ONOO(-), O(2), CO(2), and BH(4) were also modeled. The model predicted that a lower [BH(4)]/[TBP] ratio decreased NO production but increased O(2)(•-) production from eNOS. The NO and O(2)(•-) production rates were independent above 1.5μM [TBP]. The results indicate that eNOS uncoupling is a result of a decrease in [BH(4)]/[TBP] ratio, and a supplementation of BH(4) might be effective only when the [BH(4)]/[TBP] ratio increases. The results from this study will help us understand the mechanism of endothelial dysfunction.

Figure 1. eNOS structure and [BH₄]/[TBP] dependent NO and O₂^•− production

Panel A: Structure of eNOS showing the reductase and oxygenase domain with co-factors (light green), substrates L-Arginine and O₂ (yellow) and products L-citrulline (blue), NO (red) and O₂^•− (purple). Panel B: The effects of binding of reduced biopterin (BH₄) and oxidized biopterin (BH₂) to eNOS on the products of catalytic cycle of eNOS. eNOS binding of BH₄ causes NO production and that of BH₂ causes production of O₂^•−. The NO and O₂^•− formed can react with each other to form ONOO⁻, which along with O₂^•− can oxidize BH₄ to BH₃. The BH₃ formed can be oxidized to BH₂ by O₂.

Figure 2. Schematic of the reaction pathways associated with eNOS

Panel A shows the biochemical pathways of eNOS related to the production of NO by oxidation of L-Arginine upon binding of BH₄ to eNOS as well as the biochemical pathway associated with the production of O₂^•− upon binding of BH₂ to eNOS. Green represents the co-factors, grey the enzyme, orange the enzyme substrate complexes, yellow the substrates, blue, red and purple represent the products of eNOS catalysis i.e. NHA (blue), L-citrulline (blue), NO (red) and O₂^•− (purple). Panel B shows the reactions associated with NO, O₂^•− and their mutual reaction product peroxynitrite (ONOO⁻) as well the reactions associated with O₂ and CO₂ and the different forms of biopterin (BH₄, BH₃ and BH₂).

Figure 3. [BH₄]/[TBP] and [BH₄] dependent NO and O₂^•− production rate by eNOS

The biopterin ratio ([BH₄]/[TBP]) was varied from 0.99, 0.9, 0.7, 0.5, 0.25 and 0.05. Panel A, and B represents the time dependent profiles of NO production rate, and the O₂^•− production rates, respectively with respect to [BH₄]/[TBP] ratio. Panel C represents the steady state production rates of NO and O₂^•− with respect to concentration of BH₄. The concentration of total biopterin ([TBP]) was set at a constant value of 7 µM for all the different biopterin ratios simulated. The concentrations of L-Arginine, O₂, SOD, CO₂ and eNOS were set at constant values of 100µM, 140µM, 10 µM, 1.1 mM and 0.097 µM, respectively for all the cases simulated.

Figure 4. Peroxynitrite concentration profile for [BH₄]/[TBP] ratio of 0.99, 0.9, 0.7, 0.5, 0.25 and 0.05

The peroxynitrite generation was only from the eNOS produced NO and O₂^•−. The concentrations of total biopterin ([TBP]), L-Arginine, O₂, SOD, CO₂ and eNOS were 7 µM, 100µM, 140µM, 10 µM, 1.1 mM and 0.097 µM, respectively.

Figure 5. Effect of total biopterin concentration

Panel A and B show the eNOS related NO and O₂^•− production rates, respectively for [BH₄]/[TBP] ratio of 0.99, 0.9, 0.5 and 0.05. The concentrations of L-Arginine, O₂, SOD, CO₂ and eNOS were 100µM, 140µM, 10 µM, 1.1 mM and 0.097 µM, respectively.

Figure 6. Effect of eNOS concentration on the NO production rate

Panel A and B show the effect the NO production rates for biopterin ratio ([BH₄]/[TBP]) of 0.99 and 0.05, respectively. The concentrations of total biopterin ([TBP]), L-Arginine, O₂, SOD, and CO₂ were 7 µM, 100µM, 140µM, 10 µM, and 1.1 mM, respectively.

Figure 7. Effect of eNOS concentration on the O₂^•− production rate

Panel A and B show the effect the O₂^•− production rates for biopterin ratio ([BH₄]/[TBP]) of 0.99 and 0.05, respectively. The concentrations of total biopterin ([TBP]), L-Arginine, O₂, SOD, and CO₂ were 7 µM, 100µM, 140µM, 10 µM, and 1.1 mM, respectively.

Figure 8. Effects of feedback inhibition pathways for consumption of NO

Panel A shows the production rate of NO and Panel B shows the temporal NO concentration profiles. The concentrations of total biopterin ([TBP]), L-Arginine, O₂, SOD, CO₂ and eNOS were 7 µM, 100µM, 140µM, 10 µM, 1.1 mM and 0.097 µM, respectively. The biopterin ratio [BH₄]/[TBP] was 0.99.

Figure 9. Effect of incorporation of GSH in the eNOS biochemical pathway

Panel A shows the temporal variation in the GSH concentration for biopterin ratios of 0.99, 0.9, 0.7, 0.5, 0.25 and 0.05, respectively at initial GSH concentrations ([GSH]₀) of 0.1 mM (left), 1 mM (center) and 10 mM (right), respectively. Panel B shows the temporal variation in the GSSG concentration for biopterin ratios of 0.99, 0.9, 0.7, 0.5, 0.25 and 0.05, respectively at initial GSH concentrations ([GSH]₀) of 0.1 mM (left), 1 mM (center) and 10 mM (right), respectively. The concentrations of total biopterin ([TBP]), L-Arginine, O₂, SOD, CO₂, and eNOS were 7 µM, 100µM, 140µM, 10 µM, 1.1 mM and 0.097 µM, respectively.