Hydrogen peroxide (H2O2) causes oxidative stress in cells. Peroxide signals through transcription factors Nrf2 and NF-kB. By the way, Nrf2 is a leucine zipper protein which activates the promoters of genes like: NADPH quinone oxidoreductase which uses NADPH to reduce reactive quinones produced from peroxide, glutathione S-transferases which use glutathione to reduce reactive oxygen species and reverse damage to proteins, and efflux pumps which remove toxic compounds from the cell.

Hydrogen peroxide is known to inhibit the citric acid cycle and researchers have identified that the enzyme aconitase is inhibited 100% by 50uM of hydrogen peroxide. The other enzymes of the CAC are working normally at this concentration of hydrogen peroxide. Neurons are isolated and levels of NADH and CAC activity are measured.

A. How many ATP would be produced from complete catabolism of a molecule of glucose 6-phosphate in these cells with and without hydrogen peroxide? Assume the respiratory chain is functioning normally (even with peroxide) to allow for NADH to power synthesis of 2.5ATP and FADH2 to power the synthesis of 1.5ATP when oxygen is present.

B. A surprising and alarming result was obtained in these neuron cultures. When glutamate was present in the extracellular media, the amount of NADH and ATP were exactly the same in both untreated and hydrogen peroxide treated cells. The activity of glutamate dehydrogenase is shown below.

Outline the catabolism of glutamate and note the total NADH and ATP produced from 1 molecule of glutamate as it compares to 1 molecule of acetyl-CoA each being catabolized in the citric acid cycle. How would you classify glutamate dehydrogenase in terms of its enzymatic activity? What about its role in metabolism?

C. Researchers attempted to confirm their suspicion that the glutamate was serving as the energy source for neurons by depleting glucose from the media. In fact, when glucose was depleted, NADH and ATP levels were unchanged from part B. In other words, with and without hydrogen peroxide, steady state NADH and ATP levels were the same, even when no glucose was present.

Did they confirm their suspicion? Speculate on what is happening in neurons given these data.

D. Predict what you believe happens to the rates of isocitrate dehydrogenase and succinate dehydrogenase activity in neurons treated with hydrogen peroxide in the absence of glucose and presence of glutamate.

E. Higher concentrations of hydrogen peroxide (>100uM) inhibit the enzyme a-ketoglutarate dehydrogenase.

Speculate on what you believe will happen to the NADH and ATP concentrations neurons in the presence of 110uM hydrogen peroxide in the presence of glutamate and glucose.