Comparative kinetic isotope effects on first- and second-order rate constants of soybean lipoxygenase variants uncover a substrate-binding network.

Lipoxygenases are widespread enzymes found in virtually all eukaryotes, including fungi, and, more recently, in prokaryotes. These enzymes act on long-chain polyunsaturated fatty acid substrates (C18 to C20), raising questions regarding how the substrate threads its way from solvent to the active site. Herein, we report a comparison of the temperature dependence of isotope effects on first- and second-order rate constants among single-site variants of the prototypic plant enzyme soybean lipoxygenase-1 substituted at amino acid residues inferred to impact substrate binding. We created 10 protein variants including four amino acid positions, Val-750, Ile-552, Ile-839, and Trp-500, located within a previously proposed substrate portal. The conversion of these bulky hydrophobic side chains to smaller side chains is concluded to increase the mobility of flanking helices, giving rise to increased off rates for substrate dissociation from the enzyme. In this manner, we identified a specific binding network that can regulate movement of the substrate from the solvent to the active site. Taken together with our previous findings on C-H and O2 activation of soybean lipoxygenase-1, these results support the emergence of multiple complementary networks within a single protein scaffold that modulate different steps along the enzymatic reaction coordinate

Hydrostatic Pressure Studies Distinguish Global from Local Protein Motions in C-H Activation by Soybean Lipoxygenase-1.

The proposed contributions of distinct classes of local versus global protein motions during enzymatic bond making/breaking processes has been difficult to verify. We employed soybean lipoxygenase-1 as a model system to investigate the impact of high pressure at variable temperatures on the hydrogen-tunneling properties of the wild-type protein and three single-site mutants. For all variants, pressure dramatically elevates the enthalpies of activation for the C-H activation. In contrast, the primary kinetic isotope effects (KIEs) for C-H activation and their corresponding temperature dependencies remain unchanged up to ca. 700 bar. The differential impact of elevated hydrostatic pressure on the temperature dependencies of rate constants versus substrate KIEs provides direct evidence for two distinct classes of protein motions: local, isotope-dependent donor-acceptor distance-sampling modes, and a more global, isotope-independent search for productive protein conformational sub-states

Comparative kinetic isotope effects on first- and second-order rate constants of soybean lipoxygenase variants uncover a substrate-binding network.

Lipoxygenases are widespread enzymes found in virtually all eukaryotes, including fungi, and, more recently, in prokaryotes. These enzymes act on long-chain polyunsaturated fatty acid substrates (C18 to C20), raising questions regarding how the substrate threads its way from solvent to the active site. Herein, we report a comparison of the temperature dependence of isotope effects on first- and second-order rate constants among single-site variants of the prototypic plant enzyme soybean lipoxygenase-1 substituted at amino acid residues inferred to impact substrate binding. We created 10 protein variants including four amino acid positions, Val-750, Ile-552, Ile-839, and Trp-500, located within a previously proposed substrate portal. The conversion of these bulky hydrophobic side chains to smaller side chains is concluded to increase the mobility of flanking helices, giving rise to increased off rates for substrate dissociation from the enzyme. In this manner, we identified a specific "binding network" that can regulate movement of the substrate from the solvent to the active site. Taken together with our previous findings on C-H and O2 activation of soybean lipoxygenase-1, these results support the emergence of multiple complementary networks within a single protein scaffold that modulate different steps along the enzymatic reaction coordinate

Hydrostatic Pressure Studies Distinguish Global from Local Protein Motions in C−H Activation by Soybean Lipoxygenase‐1

The ability to relate a hierarchy of protein motions to function remains a compelling experimental challenge at the interface of chemistry and biology. In particular, the proposed contribution of distinctly different classes of local vs. global protein motions during enzymatic catalysis of bond making/breaking processes has been difficult to capture and verify. Herein we employ soybean lipoxygenase-1 as a model system to investigate the impact of high pressure at variable temperatures on the hydrogen tunneling properties of wild type protein and three single site mutants. For all variants, pressure dramatically elevates the experimental enthalpies of activation accompanying the C-H activation step, as predicted for non-physiological conditions that lead to impairment of a protein’s global conformational landscape. In marked contrast, the primary kinetic isotope effects for C-H activation and their corresponding temperature-dependencies remain unchanged up to ca. 700 bar. The differential impact of elevated hydrostatic pressure on the temperature dependencies of rate constants, vs. substrate kinetic isotope effects provides direct experimental verification of two classes of protein motions: local, isotope-dependent donor-acceptor distance sampling modes that are distinct from the more global, isotope independent search for productive protein conformational sub-states

Comparative kinetic isotope effects on first- and second-order rate constants of soybean lipoxygenase variants uncover a substrate-binding network.

Lipoxygenases are widespread enzymes found in virtually all eukaryotes, including fungi, and, more recently, in prokaryotes. These enzymes act on long-chain polyunsaturated fatty acid substrates (C18 to C20), raising questions regarding how the substrate threads its way from solvent to the active site. Herein, we report a comparison of the temperature dependence of isotope effects on first- and second-order rate constants among single-site variants of the prototypic plant enzyme soybean lipoxygenase-1 substituted at amino acid residues inferred to impact substrate binding. We created 10 protein variants including four amino acid positions, Val-750, Ile-552, Ile-839, and Trp-500, located within a previously proposed substrate portal. The conversion of these bulky hydrophobic side chains to smaller side chains is concluded to increase the mobility of flanking helices, giving rise to increased off rates for substrate dissociation from the enzyme. In this manner, we identified a specific "binding network" that can regulate movement of the substrate from the solvent to the active site. Taken together with our previous findings on C-H and O2 activation of soybean lipoxygenase-1, these results support the emergence of multiple complementary networks within a single protein scaffold that modulate different steps along the enzymatic reaction coordinate

The Regulatory Effects and the Signaling Pathways of Natural Bioactive Compounds on Ferroptosis

Natural bioactive compounds abundantly presented in foods and medicinal plants have recently received a remarkable attention because of their various biological activities and minimal toxicity. In recent years, many natural compounds appear to offer significant effects in the regulation of ferroptosis. Ferroptosis is the forefront of international scientific research which has been exponential growth since the term was coined. This type of regulated cell death is driven by iron-dependent phospholipid peroxidation. Recent studies have shown that numerous organ injuries and pathophysiological processes of many diseases are driven by ferroptosis, such as cancer, arteriosclerosis, neurodegenerative disease, diabetes, ischemia-reperfusion injury and acute renal failure. It is reported that the initiation and inhibition of ferroptosis plays a pivotal role in lipid peroxidation, organ damage, neurodegeneration and cancer growth and progression. Recently, many natural phytochemicals extracted from edible plants have been demonstrated to be novel ferroptosis regulators and have the potential to treat ferroptosis-related diseases. This review provides an updated overview on the role of natural bioactive compounds and the potential signaling pathways in the regulation of ferroptosis

Evaluation of TROPOMI and OMI Tropospheric NO₂ Products Using Measurements from MAX-DOAS and State-Controlled Stations in the Jiangsu Province of China

The tropospheric vertical column density of NO2 (Trop NO2 VCD) can be obtained using satellite remote sensing, but it has been discovered that the Trop NO2 VCD is affected by uncertainties such as the cloud fraction, terrain reflectivity, and aerosol optical depth. A certain error occurs in terms of data inversion accuracy, necessitating additional ground observation verification. This study uses surface NO2 mass concentrations from the China National Environmental Monitoring Center (CNEMC) sites in Jiangsu Province, China in 2019 and the Trop NO2 VCD measured by MAX-DOAS, respectively, to verify the Trop NO2 VCD product (daily and monthly average data), that comes from the TROPOspheric Monitoring Instrument (TROPOMI) and Ozone Monitoring Instrument (OMI). The results show that the spatial distributions of NO2 in TROPOMI and OMI exhibit a similar tendency and seasonality, showing the characteristics of being high in spring and winter and low in summer and autumn. On the whole, the concentration of NO2 in the south of Jiangsu Province is higher than that in the north. The Pearson correlation coefficient (r) between the monthly average TROPOMI VCD NO2 and the CNEMC NO2 mass concentration is 0.9, which is greater than the r (0.78) between OMI and CNEMC; the r (0.69) between TROPOMI and the MAX-DOAS VCD NO2 is greater than the r (0.59) between OMI and the MAX-DOAS. As such, the TROPOMI is better than the previous generation of OMI at representing the spatio-temporal distribution of NO2 in the regional scope. On the other hand, the uncertainties of the satellite products provided in this study can constrain regional air quality forecasting models and top-down emission inventory estimation