Lecture 19 - TCA Cycle/Citric Acid Cycle and Krebs Cycle

Why is FAD the electron acceptor in the 6th step of the TCA cycle?

- Bcause the free energy change of the reaction is insufficienct to reduce NAD+.

What is important in terms of the energy of Succinyl CoA?

- Is an energy-rich thioester compound (thioester bond is energy rich) --> A compound with HIGH PHOSPHORYL TRANSFER POTENTIAL.

Why is the cycle called Krebs cycle, the citric acid cycle and the TCA cycle?

- Krebs cycle is from Sir Hans Krebs - Citric acid cycle from the first compound made when acetyl CoA joins. - TCA: tricarboxylic acid cycle from the three carboxylates on citrate

What happens in the final 3 steps of the TCA cycle?

- Reaction of 4C compounds consitute the final stage of the citric acid cycle: REGENERATION OF OCALOACETATE. A methylene group (CH2) is converted into a carbonyl group (C=O) in 3 steeps: OXIDATION, HYDRATION and a second OXIDATION reaction.

What is the fourth rection of the TCA cycle?

- Second oxidative decarboxylaiton step, of a-ketoglutarate (C5) to form Succinyl CoA (C4). - This is catalyzed by the a-ketoglutarate dehydrogenase COMPLEX. - Removes CO2, adds CoA and uses NAD+ as the electron carrier. a-Ketoglutarate + CoA + NAD+ --> succinyl CoA + CO2 + NADH

What is the 5th step of the TCA cycle?

- Succinate-CoA (C4) is cleaved to form Succinat, CoA and GTP. - Cleavage of the bond is highly favorabl (DG =-3.3kJ) - THe cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate, usually GDP (Pi is added to form GTP). - This reaction is catalyzed by succinyl CoA synthesase (aka succinate thiokinase)

Outline and draw the pathway of succinyl CoA synthesase

- The mechanism of this reaction is a clear example of an energy transformation: energy inherent in the thioester molecule is TRANSFORMED into PHOSPHRYL-GROUP-TRANSFER POTENTIAL.

What is the third step of the TCA cycle?

- The thrid step is the oxidative decarbolyation of D-isocitrate (C6) to produce a-ketoglutarate (C5), CO2 and NADH. This is catalyzed by isocitrate dehydrogenase. Isocitrate + NAD+ --> a-ketoglutarate + CO2 + NADH. Generates the first high transfer potentil electron carrier, NADH in the cycle.

What is characteristic of the energy needed for the last reaction?

- Unlike the other reactions, the standard free energy of this reaction is significantly positive (+29.7 kJ mol-1). THe oxidation of malate is friven by the use of the products-oxaloacetate by citrate synthase and NADH by the electron transport chain.

How many energy equivalents are transfered in the calvin cycle? What are they transfered to?

1 hydride ions (hence size electrons) are transferred to three molecules of nicotinamide adenine dinucleotide (NAD+) 1 pair of hydrogen atoms (hence two electons) is transferred to one molecule of flavine adenin dinucleotide

What step connects the glycolytic pathway to the TCA cycle?

At the end of the glycolytic pathway pyruvate is the product. This must be converted into acetyl CoA for entry into the TCA cycle. CO2 is lost and a molecule of NADH is made.

Where is Pyruvate Dehydrogenase found in bacteria and eukaryotic cells?

Bacteria (E.coli): Cytosol and in the matrix of mitochondria. Eukaryotes: mitochondrial matrix (After it has been carried across by a membrane protein)

What is the enzyme called used in the first step of the Calvin cycle, and why?

Citrate synthase. An enzyme catalyzinf a synthetic reaction in which 2 units are joined usually WITHOUT the direct participation of ATP (or another nucleoside triphosphate)

How many subuints are in the E.coli and mammalian Pyruvate Dehydrogenase?

E.coli: 60 Mammalian: 294

What is the structure of E1 in Pyruvate Dehydrogenase

E1 is itself and tetramer of a2b2.

Where are the enzymes for the TCA cycle located in eukaryotic cells and E.coli?

EUKARYOTIC: all within the mitochondrial matrix E.COLI enzymes are in the cytosol (cytoplasm) and in mitochondrial matix.

What does FAD stand for? How many energy equivalents can it transfer?

Flavin adenine dinucleotide Can carry 2 H+ and 2e- (two protons and two electrons) in its fully reduced form

What is the purpose of the TCA cycle?

Harvesting of high-energy electrons in co-factors from carbon fuels, to synthesize ATP.

Who came up with the underlying theory of the cycle?

In 1937 Sir Hans Krebs based in the University of Sheffield summarised the known work on a cycle of reactions that included citric acid and that could explain the complete oxidation of sugars. This was based on many scientists previous work through the 1930s.

In E.coli what does the Succinyl CoA synthesase generate?

In E. coli the SCS enzyme can generate either ATP or GTP.

How does pyruvate enter the mitochondrial matrix?

In eukaryotic cells pyruvate is generated outside the mitochondria from glycolysis and enters the mitochondria via a pyruvate/H+ symporter (this means the pyruvate and H+ enter together) and consumes energy.

In mammalian cells where is fumerase found, and what is the enzyme invovlved in?

In mammalian cells the enzyme is found both in the mitochondrion and in the cytoplasm, where it is involved in amino acid catabolism and the urea cycle. Also involved in the TCA cycle in the mitochondrial matrix.

In what type of reactions if FAD used?

In oxidation reactions. Where an enzyme catalyzes the oxidation of a compmound, it will subsequently reduce FAD to FADH2.

What is the importance of Pyruvate Dehydrogenase ?

It is a very important enzyme as it converts the products of sugar metabolism to the acetyl CoA needed for the TCA cycle

Why is Ubiquinone found in the inner membrane of the mitochondria?

It is not soluble in water because of long side chain, so remins in mitochondrial inner membrane. It is membrane bound.

Is the first step of the Krebs cycle favorable, and if so why?

It is. The standard DG is positive but the DG of the reaction is negative (influenced by product/reactant concentration). Citrate is being consumed in the cycle, pulling the equilibrium to the right. The HYDROLYSIS of the thioester bond between CoA and S powers the reaction (this is a high energy bond)

Outline the structure of FAD. Why is the cofactor easy to reduce?

Large hande = the nezyme holds onto tightly, here it is adenine Then open sugar chain = tibitol = sugar alcohol = links to the nitrogen group at flavin. Flavin = 3 rings, so lots of free electrons, so easy to reduce.

Where is Malate dehydrogenase found in cells?

Malate dehydrogenase is found in both the mitochondrion and the cytoplasm

Where does the TCA cycle occur in eukaryotic cells?

Mitochondrial matrix

Is the fourth step of the KRebs cycle favorable?

Yes. Relatively large negative DG.

What is an alternative name for a-ketoglurate?

2-oxoglutarate

How many different forms does Flavin adenin dinucleotide exist in?

3

How many oxidation-reduction steps occur in Krebs cycle?

4

Where does acetyl CoA from B-oxidation come from, before entering the TCA cycle?

Acetyl CoA from β-oxidation is generated within the mitochondrial matrix. Has already entered the matrix with___protein___.

What is the net reaction of the TCA cycle? Summarize the cycle.

Acetyle CoA + 3NAD+ + FAD + ADP + Pi + 2H2O --> 2 CO2 + 3NADH + FADH2 + ATP + 2H+ + COA 1.) Two carbon atoms eneter the cycle in the condensation of an acetyl unit (from acetyle CoA) with oxaloacetate. Two carbon atoms leave the cycle in the form o fCO2 in the succesive decarboxylations catalyzed by isocitrate dehydrogranse and a-ketoglutarate dehygrogenase. 2. Four pais of hydrogen atoms leave the cycle in four oxidation reactions. Two NAD+ molecules are reduced in the oxidative decarboxylations of isocitrate and a-ketoglutarate, one FAD molecule is reduced in the oxidation of succinate, and one NAD+ is reduced in the oxidation of malate. One NAD+ molecule is reduced in the oxidative decarboxylaiton of pyruvate to form acetyle COA. 3. One compound with high phosphoryl transfer potential, usually ATP is generated form the cleavage of the thioester linkage in succinyl CoA. 4. Two water molecules are consumed: one in the synthesis of citrate by the hydrolysis of citryl CoA and the other in the hydration of fumerate.

How are reducing equivalents from glycolysis transported into the cell?

NADH from glycolysis most be transported into the mitochondrial matrix to contribute to the electron transport chain. Malate = serves as a reducing agent, NADH DOES NOT MOVE THROUGH THE MEMBRANE. Has the net effect of forming NADH on the inside of the cell.

Does the citric acid cycle generated ATP or use oxygen?

NO, instead the cycle removes electrons from acetyl CoA and uses these electrons to reduce NAD+ and FAD to form NADH and FADH2.

Is the second step of the Krebs cycle favorable?

No. Has a positive standard DG +6.5 However it occurs because the iso citrate is being pulled through the cycle, changing the DG of the reaciton.

How much ATP is generated from one acetyl CoA going through the Krebd cycle?

One molecule directly synthesized. The energy equivalents are used to pump protons across the mitochondial inner membrane . These electron carriers yield nine molecules of ATP when they are oxidiced by O2 in oxidative phosphorylation.

What is the first step of the TCA cycle? Explain and provide the enzyme.

Oxaloacetate reacts with acetyle CoA and H2O to yield citrate and CoA: - CONDENSATION of a 4C unit (oxaloacetate) and a 2C unit, the acetyle group of the acetyle CoA --> to form an aldol. -Followed by hydrolysis to form citrate and CoA. The enzyme used is called CITRATE SYNTHASE.

What enzyme catalyzes the oxidative decarboxylaiton of pyruvate?

Pyruvate Dehydrogenase catalyzes oxidative decarboxylation of pyruvate, to form acetyl-CoA. Pyruvate + CoA + NAD+ --> acetyle CoA + CO2 + NADH + H+ This reaction is irreversible. This produces CO2 and captures high-transfer potential electrons in the form of NADH.

What happens after pyruvate enters the mitochondrial matrix in E.coli? Explain the enzyme and the chemical equation.

Pyruvate Dehydrogenase catalyzes oxidative decarboxylation of pyruvate, to form acetyl-CoA. PDH is the in MITOCHONDRIAL MATRIX. Pyruvate + CoA + NAD+ --> acetyle CoA + CO2 + NADH + H+ This reaction is irreversible. This produces CO2 and captures high-transfer potential electrons in the form of NADH.

What is the 6th step of the TCA cycle?

Succinate is OXIDIZED to fumarate by succinate dehydrogenase. The hydrogen accepted is FAD rather than NAD+, which is used in the other 3 oxidation reactions in the cycle.

What is the 7th steo of the TCA cycle?

THe hydration of fumerate to form L-malate. - Fumerase catalyzes the addition of water across the double bond.

What is the fate of the electrons accepted by FAD in the 6th step of the TCA cycle?

The FADH2 is tightly bound to the enzyme (succinate dehydrogenase which is in the inner membrane of the mitochondria) The electrons captured in the FADH2 are transferred to a lipid soluble electron carrier (THROUGH ION SULFATE CLUSTERS IN THE PROTEIN, through to a lipid electron carrier) called ubiquinone that always stays in the membrane. This is ONE OF THE ROUTES OF ENERGY EQUIVALENTS (ELECTRONS) into the electron transport chain

What happens to the NADH and FADH2 produced in the TCA cycle?

The NADH and FADH2 are oxidised at the electron transport chain which we will look at in another lecture.

Define TCA cycle

The citric acid cycle (CAC) - also known as the tricarboxylic acid (TCA) cycle or the Krebs cycle[1][2] - is a series of chemical reactions used by all aerobic organisms to release stored energy through the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins into carbon dioxide and chemical energy in the form of adenosine triphosphate (ATP).

What is the 8th and final step of the TCA cycle?

The final step of the citric acid cycle recreates oxaloacetate, transferring electrons to NADH in the process. Malate dehydrogenase catalyzes the reaction. Malate + NAD+ --> oxaloacetate + NADH + H+.

What is the second step of of the Krebs cycle?

The hydroxyl group of citrat is not properly located in the citrate molecule for the OXIDATIVE DECARBOXYLATION that follows. Thus citrate is isomerized into D-isocitrate to enable the 6C unit to undergo oxidative decarboxylation. The isomerization is accomplised by a dehydration step follwed by a hydration step. The result is an interchange of H and an OH. The enzyme is called ACONITASE (because cis-aconitate is an intermediate)

What other pathways does the 3rd step of the Calvin cycle link to?

The α-ketoglutarate (also known as 2-oxoglutarate) is a link to NITROGEN METABOLISM. GLUTAMATE enters the TCA cycle as a-ketoglutarate after removal of a nitrogen group from the keto group: >>>>The amino acid glutamate is derived from α-ketoglutarate by addition of nitrogen to the keto group. Transaminases (enzyme used in the synthesis of AA) can also feed the TCA cycle with α-ketoglutarate by removal of the NH2

Discuss the structure of Pyruvate Dehydrogenase in E.coli and mammalian cells.

There are THREE subunits: E1, E2, and E3 E.COLI: E1: 24, E2: 24, E3: 12 MAMMALIAN: E1: 30, E2: 60, E3: 12 + 12 COPIED OF A BINDING PROTEIN THAT LNK E2 TO E3.

What do all fuel components have in common, in terms of pathway?

They are all ultimatley metabolized to acetyl CoA or components of the citric acid cycle.

What are the reactants and products of the TCA cycle?

This cycle takes acetyl CoA generated by the breakdown of sugars, fats (lipids) and proteins to generate GTP and reduced cofactors NADH and FADH2 Overall 1 molecule of acetyl CoA are converted to 2 molecules of CO2 and 3 NADH, 1 FADH2 and 1 GTP.

Where and how is GTP converted to ATP?

This is converted to ATP in mitochondria by the enzyme nucleoside diphosphate kinase.

Where is succinate dehydrogenase located? Why is this useful/related to function?

This is an enzyme complex that has part of it in the inner membrane (one subunit with alfa helizes that are hydrophobic that interact with the lipids in the membrane so permanently embedded) of the mitochondrion. It links its citric acid cycle task directly to the electron transport chain. It first extracts hydrogen atoms from succinate, transferring them to the carrier FAD. With the help of several ironsulfur clusters and a heme, these are then transferred to the mobile carrier ubiquinone, for transport to cytochrome bc1.

What is the difference between B-oxidation and glycolysis in terms of energy generation?

Unlike glycolysis, there is no net ATP generation in the actual pathway. The products from β-oxidation are reduced cofactors NADH and FADH2 and acetyl CoA. These need to go through other pathways to yield their energy. But ultimately fatty acids yield more energy per molecule than glucose.

Decribe the enzyme complex used in the 4th reaction of TCA cycle.

α-ketoglutarate dehydrogenase An organized assembly of 3 kinds of enzyme that is homologuous to the pyruvate dehygrogenase complex. In fact the E3 component is identical in both enzymes.