Tca Cycle

Front 1

Step 1: What is it? What kind of bond formation is formed? Is it reversible? What is important about this reaction? How is it regulated? How is carbon count changed? Are there any side products?

Back 1

It is the formation of citrate from condensation of acetyl-CoA with oxaloacetate, catalyzed by citrate synthase. This is the only reaction that forms a C-C single bond. The reaction is highly favorable/irreversible. This reaction is one of the regulatory and rate-limiting steps. Its activity depends largely on [oxaloacetate], and it is inhibited by buildup of other TCA intermediates. 4 Carbon OAA adds to 2 carbon acetyl CoA to make 6 carbon citrate.

Front 2

Citrate Synthase: What is special about this enzyme with regards to the first step? What is the benefit of having a closed conformation?

Back 2

Citrate synthase has two domains for binding its two substrates. It has its open conformation, where it doesn’t have a binding site for acetyl-CoA, but it can bind to OAA. When OAA binds, it induces a large conformational change (from open to closed conformation of citrate synthase) in one of the domains, creating a binding site for the second substrate, acetyl CoA. The closed conformation avoids unnecessary and unproductive hydrolysis for acetyl CoA thioester.

Front 3

Step 2: What is it? How does water play a role in this step? Is it reversible? How is it regulated? How is carbon count changed?Are there any side products?

Back 3

The enzyme aconitase catalyzes the reversible transformation of citrate to isocitrate through the intermediate formation of cis-aconitate. Elimination of water from citrate gives a cis C=C double bond in cis-aconitate, but addition of it (which is stereospecific) will lead to isocitrate. The reaction is close to equilibrium, so product concentration is kept low to pull reaction forward. No change in carbons.

Front 4

Stereospecificity of Aconitase: How is it stereospecific?

Back 4

Only one isomer of isocitrate is produced by aconitase, which is distinguished by its three-point attachment to the active site.

Front 5

Step 3: What is it? Is the reaction reversible? How is it regulated? How is carbon count changed? Are there any side products? What is important about one of its products?

Back 5

Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to form alpha-ketoglutarate. In the oxidative decarboxylation, the alcohol is oxidized to a ketone, a carbon is lost as CO2 (it did not come from acetyl-CoA but from OAA), and NADH is generated during oxidation. The reaction is highly favorable/irreversible. It is regulated by product inhibition and ATP, ADP, and Ca2+. Alpha-ketoglutarate is an important branch point for amino acid metabolism. Change from 6C to 5C.

Front 6

Isocitrate Dehydrogenase: What is specialabout this enzyme?

Back 6

There are two forms of this enzyme in all cells: The NAP-dependent enzyme exists in the mitochondria. The other NADP-dependent enzyme is found in both cytosol (where it is used as a cofactor) and mitochondria.

Front 7

Step 4: What is it? How is carbon count changed? What happens after two turns of the cycle? What is special about the product? How is carbon count changed?Are there any side products? What is important about one of its products?

Back 7

Another oxidative decarboxylation occurs, where alpha-ketoglutarate is converted to succinyl-CoA and CO2 by the alpha-ketoglutarate dehydrogenase complex. An NAD+ serves as an electron acceptor, and CoA acts as the carrier of the succinyl group. The carbon lost did not come from oxaloacetate. After two turns of the cycle, there is a net full oxidation of all carbons of glucose. Succinyl-CoA is another higher-energy thioester bond where energy from CO2 oxidation is stored. It also communicates flow to the start of the cycle. Change from 5 carbons to 4 carbons.

Front 8

Alpha-ketoglutarate dehydrogenase complex: What is its difference to pyruvate dehydrogenase complex?

Back 8

Complexes are similar to each other with same coenzymes and identical mechanisms, but their active sites are different to accommodate different-sized substrates.

Front 9

Step 5: What is it? What is substrate-level phosphorylation in this step? Is this reaction reversible? Are ATP and GTP different in terms of energy? How is carbon count changed? Are there any side products?

Back 9

High-energy thioester bond is broken to drive the synthesis of a phosphoanhydride bond in GTP (that can be converted to ATP in a reversible reaction), forming succinate through the enzyme succinyl-CoA synthetase. The formation of ATP/GTP at expense of energy released from oxidative decarboxylation of alpha-ketoglutarate is a substrate-level phosphorylation (similar to process in glycolysis). Reaction is close to equilibrium, so product concentration is kept low to pull reaction forward. ATP and GTP are energetically equivalent. No change in carbons.

Front 10

Step 6: What is it? Is the reaction reversible? How is the carbon count changed? Are there any side products?

Back 10

Succinate formed from succinyl-CoA is oxidized to fumarate by flavoprotein succinate dehydrogenase. Reaction is near equilibrium so product concentration is kept low to pull forward. No change in carbons.

Front 11

Step 6 Enzymes and Coenzymes: Where is Succinate Dehydrogenase located? What is special about it? What is special about FADH2?

Back 11

Succinate Dehydrogenase is bound to mitochondrial inner membrane; it is part of complex II in the electron transport chain. FADH2 provides reducing power for reduction of alkane to alkene. Nochange in carbons.

Front 12

Step 7: What is it? What is its transition state? Is the enzyme stereospecific? Is the reaction reversible? How is the carbon count changed? Are there any side products?

Back 12

Fumerate is hydrated across a double bond to malate, which is catalyzed by fumarase. The transition state is a carbanion. The OH- adds to fumarate. Then H+ adds to the carbanion. Since fumarase cannot distinguish between inner carbons of fumarate, either can gain OH-. The enzyme is stereospecific. The reaction is near equilibrium, so the product concentration is kept low to pull the reaction forward.

Front 13

Step 8: What is it? What is produced by this step that is unique to it? Is the reaction reversible? How is the carbon count changed? Are there any side products?

Back 13

This is the final step of the cycle. Malate dehydrogenase catalyzes the oxidation of malate to oxaloacetate. The enzyme is linked to an NAD+ that is reduced to NADH. It regenerates oxaloacetate for citrate synthase. This reaction is highly unfavorable, so oxaloacetate (the product) is kept very low by citrate synthase to pull the reaction forward. No change in carbons.

Front 14

Net Results of TCA Cycle: What are the net results regarding carbon atoms, energy change, and main product? What is the net reaction equation?

Back 14

Two carbon atoms emerged from the cycle in the form of CO2 from oxidation of isocitrate and alpha-ketoglutarate. These carbons are not the same carbons that entered in form of acetyl coA. The energy released from these oxidations was conserved in reduction of three NAD+ and one FAD+ and released in form of one ATP/GTP. At the end of the cycle, one oxaloacetate was regenerated. Four oxidation steps in cycle provide large flow of electrons into respiratory chain via NADH and FADH2 that leads to lots of ATP molecules generated during oxidative phosphorylation. Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H20 —> 2CO2 + 3NADH + FADH2 + GTP + coA + 3H+