Browsing by Subject "Enzyme"

Electron bifurcation oxidizes a two-electron donor, using the two electrons to reduce high- and low-potential acceptors. Thus, one electron may move thermodynamically uphill, being kinetically coupled to the downhill flow of the other electron. Electron bifurcation in nature is often reversible (∆G ≈ 0) so minimal free energy is dissipated, and the reaction occurs at minimal overpotential. Thus, electron bifurcation is a compelling target for bioinspired catalysis and/or nanoscale device design.We formulate a general theory of the electron bifurcation process, using a many-electron hopping kinetics model with hopping rate constants estimated with thermally activated electron tunneling theory. We conclude that efficient and reversible electron bifurcation requires only a conserved redox potential (free energy) landscape, with steep redox potential gradients in the high- and low-potential branches (the reversible EB scheme). This energy landscape naturally builds up electron and hole populations near the bifurcating two-electron cofactor in the high- and low-potential branches, respectively, thus disfavoring short-circuiting electron-hole combination. The reversible EB scheme suppresses short-circuiting reactions by erecting a Boltzmann penalty against redox states of the enzyme that may short-circuit, and largely accounts for short-circuit insulation in complex III of the electron transport chain, although our model does not uniquely account for the slow short-circuit turnover with an inhibited low-potential branch. For electron bifurcating enzymes that reduce the low-potential substrate directly (such as bifurcating electron transfer flavoproteins), we hypothesize that a downward shift in redox potential of the low-potential substrate upon binding to the bifurcating enzyme (similar to the iron protein bound to nitrogenase) could explain how the requisite steep redox potential gradient is achieved without housing a series of redox cofactors in the low-potential branch. Electron bifurcating enzymes in nature are often found with bifurcating cofactors (for example quinones and flavins) with inverted reduction potentials (i.e., the first reduction potential lower than the second). We derive a free energy decomposition scheme for the half-reactions of a two electron species from quantum chemical calculations to find physical and chemical factors that determine whether the reduction potentials are inverted. Remarkably, two electron species such as quinones and flavins can exhibit normally-ordered or inverted reduction potentials depending on the protein environment. Using our energy decomposition scheme and an estimate of a quinone Pourbaix diagram under continuum mean-field environments with varying electrostatic permittivity, we conclude that the proton transfer events that often accompany reduction in addition to the electrostatic interactions of charged species may have a significant impact on the invertedness of two-electron compounds. Thus, we hypothesize that electrostatic interactions (including the self-interaction of a charged semiquinone) may principally explain the ability of flavins and quinones to change the order of their first and second reduction potentials so profoundly. Future studies will be required to test this hypothesis. In addition, we show that efficient and reversible electron bifurcation is possible with normally ordered potentials at the bifurcating cofactor, provided the absolute value of the difference between the first and second reduction potentials is large (on the order of the redox potential span of the high- and low-potential branches in the reversible EB scheme). This finding has implications for synthetic electron bifurcation, as engineering redox active catalytic sites with strongly normally ordered potentials seems more straightforward than sites with strongly inverted potentials. Finally, we describe kinetics schemes for thermodynamically irreversible electron bifurcation that rely on disequilibrium populations of electrons within the high potential branch (irreversible confurcation is possible with a disequilibrium population of holes within the low-potential branch). Although these schemes are yet only hypothesized, these schemes allow orders-of-magnitude regulation of the bifurcating turnover rate with the redox poise of the two-electron donor (including faster turnover than in the reversible EB scheme), with kinetics fit by a generalized Shockley ideal diode equation.

Tumor necrosis factor  (TNF) has positive and negative roles in human disease. In certain cancers, TNF is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNF antibodies control inflammation in most patients, but these benefits are offset by cost and tachyphylaxis that develops during chronic treatment. Transforming growth factor beta-activated kinase 1 (TAK1) acts as a key mediator between survival and cell death in TNF-mediated signaling, uniquely providing a drug development opportunity for cancer and autoimmunity. Takinib is a potent and selective TAK1 inhibitor (IC50 9.5nM) that induces apoptosis in a TNF-dependent manner in cell models of metastatic breast cancer and rheumatoid arthritis. The mechanisms underlying this specificity were revealed in enzymatic and co-crystallization studies. These data show that Takinib targets the kinase in the DFG-in conformation and forms direct and water-mediated hydrogen bonds with catalytic residues. Mechanistic studies of TAK1 autophosphorylation demonstrated a substrate-like intermolecular mechanism, during which Takinib treatment slows down the rate-limiting step. Overall, our data show Takinib is an attractive starting point for the development of inhibitors that greatly sensitize cells to TNF-induced cell death, broadening the therapeutic efficacy of TNF for cancer and autoimmune disease.

Millions of people around the world eventually develop or currently suffer from some form of cancer. According to the American Cancer Society, 1.6 million new cases of cancer were predicted in the United States alone.One of the main reasons for this high mortality rate is that cancer can be an exceedingly difficult disease to treat. In response to the need for better drugs, enzymatically-activated Cu prochelators (GluBro and GluTCarb) have been developed to specifically target cancer cells and create cytotoxic complexes within tumor tissue. The experiments performed herein suggest both GluBro and GluTCarb demonstrate cancer selective cellular activity dependent on the presence of Cu and the enzyme gamma-glutamyltransferase