Question

1. In an experiment, you grow E. coli cells in the lab at different temperatures. After the growth, you isolate their cell membranes, perform complete hydrolysis of their cell membranes and isolate and quantify the percentages of different fatty acids in your final sample. How would you expect the fraction of myristic acid to change as you increase the temperature?  

2. Succinyl-CoA is a negative regulator of α-ketoglutarate-dehydrogenase complex. Explain why this makes sense, and propose a possible mechanism for how this molecule inhibits citrate synthase. What other step of the citric acid cycle is inhibited by succinyl- CoA, and how do you expect that inhibition mechanism to be similar/different?

3. In gluconeogenesis, the pyruvate to phosphoenolpyruvate is one of the bypass steps that is different from the reverse reaction in glycolysis due to the irreversible nature of pyruvate kinase. There are two pathways for this bypass reaction that differ in the location where the final PEP product is synthesized – either in the mitochondria or in the cytoplasm. Assume that your cell is undergoing gluconeogenesis using pyruvate derived from the deamination of serine by the enzyme serine dehydratase:

L-serine → Pyruvate + NH3

Which of the two pathways of phosphoenolpyruvate synthesis will be used and why? What are the intermediate steps for this reaction, and where do they occur?

Answer 1

1. The transition temperature is a function of the membrane lipid composition and in organisms deficient in cholesterol, mainly depends on the fatty acid composition of the membrane lipids. Phospholipids that contain unsaturated fatty acids (UFAs) have much lower transition temperatures than those lipids made of saturated fatty acids (SFAs).  The E. coli cells have mechanisms to change the relative amounts of saturated fats and unsaturated fats in their cell membranes. The higher the cell's growth temperature, the less unsaturated material; the lower the temperature the more unsaturated material. This turns out to be essential for survival. Myristic acid is a 14-carbon, unsaturated fatty acid. Therefore higher the temperature of E. coli cell's growth less the fraction of myristic acid.

2. Succinyl-CoA is a negative regulator of α-ketoglutarate-dehydrogenase complex. Several allosteric inhibitors such as NADH and ATP (both of which are products of the Citric acid cycle) that control the activity of citrate synthase. These two molecules bind allosterically to citrate synthase and decrease its affinity for it substrates. Succinyl-coA is a product of a later step in the Citric acid cycle and it accumulation in the mitochondrial matrix indicates an abundance of electron carriers (in the form of NADH and FADH2) and hence free energy (in the form of ATP) in the cell, suggesting that catabolism can be halted. Succinyl-coA, regulates citrate synthase through competitive inhibition by binding to its active site, thus blocking access of the substrate to the binding site. Thus, succinyl-coA serves as a competitive feedback inhibitor of citrate synthase by inhibiting over catalysis of the citric acid cycle and stopping further catabolism.

3. The majority of the enzymes responsible for gluconeogenesis are found in the cytoplasm. A few exceptions are mitochondrial pyruvate carboxylase and phosphoenolpyrvate carboxykinase (animals).

Synthesis of serine

The intermediates in the pathways of gluconeogenesis are involved in the synthesis of serine, either from glucose via glycolysis or from the triose phosphate pool, where carbon is generated from citric acid cycle intermediates.