Biochem Chapter #16 (The Citric Acid Cycle) Review

Small

Only a _________ amount of energy available in glucose is captured in glycolysis

CO2 and H2O

The full oxidation of glucose produces a significant amount of energy compared to the anaerobic oxidation of glucose in glycolysis. What is glucose eventually reduced to?

Cellular respiration

- Set of pathways that uses oxygen as a final electron acceptor to produce ATP

This is the process in which cells consume oxygen and produce carbon dioxide. This provides more ATP from glucose than glycolysis itself. This also captures energy stored in lipids and amino acids. The three major stages of this are acetyl CoA production, acetyl CoA oxidation, and electron transfer and oxidative phosphorylation

Acetyl-CoA, some ATP, and some NADH

What is the generated during the first stage of respiration where pyruvate is used by the pyruvate dehydrogenase complex?

1/3

During stage 1 of respiration, carbohydrates release what fraction of total potential CO2?

Acetyl-CoA

What is produced by the pyruvate dehydrogenase complex and is used in the TCA cycle which is catalyzed by enzymes to produce more NADH and FADH2

NADH, FADH2, and 1 GTP

- Remaining carbon atoms from carbohydrates, amino acids, and fatty acids are released during stage 2

What are the products of stage 2 of respiration where acetyl-CoA is oxidized?

NADH and FADH2

What two electron carriers are used in the oxidative phosphorylation mechanism for the electron transport chain to produce ATP?

Oxidative phosphorylation (electron transport chain)

This stage of respiration generates the vast majority of ATP during catabolism using the reduced electron carriers NADH and FADH2 to produce a proton gradient that drives ATP formation. This occurs in the mitochondria

Cytoplasm

Glycolysis occurs in the __________

Mitochondrial matrix

- Succinate dehydrogenase occurs in the inner membrane

Citric acid cycle occurs in the ___________ _________

Mitochondrial inner membrane

Oxidative phosphorylation (ETC) occurs in the __________ _________ ________

TCA cycle

What process accomplishes the oxidation of acetyl-CoA?

Mitochondrial matrix

Pyruvate dehydrogenase occurs in the __________ ________

Pyruvate

The conversion of __________ to acetyl-CoA is an oxidative decarboxylation where the first carbons of glucose are fully oxidized. This is catalyzed by the pyruvate dehydrogenase complex which requires 5 coenzymes

Pyruvate dehydrogenase complex

This catalyzes the conversion of pyruvate to acetyl-CoA and occurs in the mitochondrial matrix (a favorable reaction). This is the intermediate step between glycolysis and the TCA cycle and is a regulator for TCA. This is composed of three individual reaction complexes

TPP, lipoyllysine, and FAD

These are the three prosthetic groups of the 5 coenzymes that are required for catalysis by the pyruvate dehydrogenase complex

NAD+ and CoA-SH

These are the two co-substrates of the 5 coenzymes required for catalysis by the pyruvate dehydrogenase complex

Coenzyme A (CoA)

- Acetyl CoA has an adenine ring which has a high absorption at 260 nm

Coenzymes are not a permanent part of the enzymes' structure, so they associate, fulfill a function, and dissociate. The function of this coenzyme is to accept and carry acetyl groups. This contains a nucleotide, a pantothenic acid, and beta-mercaptoethylamine that has an active thiol group

Lipoic acid

Prosthetic groups are strongly bound to the protein, so the __________ _________ group of lipoate is covalently linked to the enzyme through an amide linkage on the E2 substrate via a lysine residue

- Pyruvate dehydrogenase (E1)

- Dihydrolipoyl transacetylase (E2)

- Dihydrolipoyl dehydrogenase (E3)

The pyruvate dehydrogenase complex is a large (up to 10MDa) multienzyme complex. What are the three components?

The short distance between catalytic sites allows channeling of substrates from one catalytic site to another; channeling minimizes side reactions. The regulation of activity of one subunit affects the entire complex

What are the advantages of multienzyme complexes?

Cryoelectronmicroscopy

- Samples in near-native frozen hydrated state, low temperature protects against radiation damage, electrons have smaller de Broglie wavelength and produce high-resolution images

How was the structure of the pyruvate dehydrogenase complex discovered? This was how they found out E1 had 30 subunits, E2 had 60 subunits, and E3 had 12 subunits

In E1, pyruvate is present and TPP carries an acetyl group to lipoate. In E2, acetyl CoA is formed. Then electron transport between E2 and E3 reduces NAD+ to NADH. Pyruvate dehydrogenase complex produces acetyl Co-A and NADH. Acetyl CoA enters the TCA cycle and NADH goes to ETC

Describe the overall reaction of the pyruvate dehydrogenase complex

E1: Step 1 - decarboxylation of pyruvate to an aldehyde

E1: Step 2 - oxidation of aldehyde to a carboxylic acid. electrons reduce lipoamide and form a thioester

E2: Step 3 - formation of acetyl-CoA (product 1)

E3: Step 4 - Reoxidation of the lipoamide cofactor

E3: Step 5 - REgeneration of the oxidized FAD cofactor, forming NADH (product 2)

What is the sequence of events in oxidative decarboxylation of pyruvate for each of the three enzymes?

3 NADH, 1 FADH2, 1 GTP

- NADH and FADH2 goes to ETC

- TCA cycle produces citrate which is part of the cycle

The citric acid (TCA) cycle oxidizes acetyl-CoA to produce what components (and how many) that can be converted into ATP?

Step 1: C-C bond formation between acetate (2C) and oxaloacetate (4C) to make citrate (6C)

Step 2: Isomerization via dehydration/rehydration

Steps 3-4: Oxidative decarboxylations to give 2 NADH

Step 5: Substrate-level phosphorylation to give GTP

Step 6: Dehydrogenation to give FADH2

Step 7: Hydration

Step 8: Dehydrogenation to give NADH

- One turn of the TCA cycle releases CO2 as it is produced in steps 3 and 4

What are the 8 steps of the citric acid cycle?

FAD

In the TCA cycle, succinate dehydrogenase uses __________ as an intermediate electron carrier

Citrate synthase

- Functions as an induced fit mechanism where oxaloacetate binds and then acetyl-CoA binds. This is a ternary mechanism with 3 parts (acetyl group bonds to oxaloacetate which binds to acetyl-CoA

This accomplishes the condensation of acetyl-CoA and oxaloacetate to form citrate. This is the only reaction with C-C bond formation, and it uses acid/base catalysis. This is the rate limiting step of the TCA cycle and activity largely depends on oxaloacetate concentration. This is highly thermodynamically favorable/irreversible and is regulated by substrate availability and product inhibition

Open conformation

A conformational change occurs upon binding oxaloacetate for the induced fit in citrate synthase. This conformation is where the free enzyme does not have a binding site for acetyl-CoA.

Closed conformation

A conformational change occurs upon binding oxaloacetate for the induced fit in citrate synthase. This conformation is where the binding of oxaloacetate creates binding for acetyl-CoA and the reactive carbanion is protected

Histidine and aspartate

Acid/base catalysis for the mechanism of citrate synthase is allowed by the binding of what two residues to the enzyme? After this, the methyl group of acetyl-CoA reacts with oxaloacetate

Acetyl-CoA attached to oxaloacetate comes off, stabilizes, and is released. Release happens through a hydrolysis reaction

During the hydrolysis of thioester in the mechanism of citrate synthase, what happens to the acetyl-CoA that was attached to oxaloacetate?

Acid/Base: The thioester linkage in acetyl-CoA activates the methyl hydrogens. Aspartate abstracts a proton from the methyl group, forming an enolate intermediate. The intermediate is stabilized by hydrogen bonding to and/or protonation by histidine

Hydrolysis: The thioester is subsequently hydrolyzed, regenerating CoA-SH and producing citrate

The mechanism of citrate synthase deals with acid/base catalysis and the hydrolysis of a thioester. Describe these two mechanisms used to produce citrate

Aconitase

This enzyme accomplishes the isomerization of citrate to isocitrate through dehydration and rehydration. Citrate is a 3° alcohol, which is a poor substrate for oxidation, but isocitrate is a 2° alcohol which is a good substrate for oxidation. This enzyme is a hydratase that eliminates water from citrate to give a cis C=C bond to form cis-aconitate. Next this adds water to cis-aconitate and is stereospecific to form isocitrate. This is thermodynamically unfavorable/reversible, so product concentration is kept low to pull the reaction forward.

Iron-Sulfur center

In aconitase, the water removal from citrate and subsequent addition to cis-aconitate are catalyzed by the ___________-__________ _________ which is sensitive to oxidative stress. These are involved in redox reactions and may take up one electron. In this case, it forms temporary bonds with citrate to catalyze hydration

Isocitrate dehydrogenase

Oxidative decarboxylation by this enzyme in the TCA cycle allows the priming of isocitrate for ixidation so that NAD(P)+ can be hydrogenated to form NAD(P)H. This is completed by a decarboxylation reaction to form alpha-ketoglutarate. The isozymes are specific for NADP+ (cytosolic) or NAD+ (mitochondrial), and the decarboxylation releases CO2. This is a highly favorable/irreversible reaction and is regulated by ATP concentration

1. Isocitrate is oxidized by hydride transfer to NAD+ or NADP+ to form oxalosuccinate. This is completed by the isocitrate dehydrogenase enzyme

2. Decarboxylation is facilitated by electron withdrawal from the adjacent carbonyl and coordinated Mn2++

3. Rearrangement of the enol intermediate generates alpha-ketoglutarate

What are the three steps of oxidative decarboxylation by isocitrate dehydrogenase?

Manganese

What ion is present in oxalosuccinate which distributes the charge

Alpha-ketoglutarate dehydrogenase complex

This enzyme accomplishes the final oxidative decarboxylation reaction of the TCA cycle where alpha-ketoglutarate, in the presence of CoA-SH and NAD+, becomes succinyl-CoA and releases CO2. This is a highly favorable reaction with a large, negative DG value. This is a similar complex compared to pyruvate dehydrogenase because it uses the same coenzymes and has identical mechanisms. However, the active sites are different to accommodate different-sized substrates

Branched chain amino acid (BCA Complex) oxidation enzyme

This enzyme oxidizes isoleucine, leucine, and valine which require specific E1 structures for binding

Alpha-ketoglutarate dehydrogenase

This is the last oxidative decarboxylation that accomplishes the net full oxidation of all carbons of glucose after two turns of the cycle. This produces succinyl-CoA which is another higher-energy thioseter bond. This is highly thermodynamically favorable/irreversible reaction that is regulated by product inhibition. This is the last reaction that produces CO2 where carbon atoms are lost in the second turn of the CAC

`Succinyl-CoA

All CO2 generated during the CAC is produced is produced before ____________-___________ is made. Both CO2 molecules lost were present on the oxaloacetate used to begin the cycle

Synthases

These enzymes catalyze condensation reactions in which no nucleotide triphosphate is required as an energy source

Synthetase

These enzymes catalyze condensation reactions that use ATP or another nucleotide triphosphate as a source of energy for the synthetic reaction

GTP

This direct source of energy is generated through a thioester by the substrate-level phosphorylation accomplished by succinyl-CoA synthetase

Succinyl-CoA synthetase

- The energy of thioester allows for incorporation of inorganic phosphate

- Goes through a phospho-enzyme intermediate

This enzyme uses the nucleotide GDP in addition to inorganic phosphate to form GTP and release CoA-SH through the conversion of succinyl-CoA to succinate. This is a slightly thermodynamically favorable/reversible reaction, but not as favorable as the alpha-ketoglutarate dehydrogenase reaction. Therefore, the product concentration is kept low to pull forward. This accomplishes substrate-level phosphorylation to produce GTP which can be converted to ATP

Succinyl-CoA enters succinyl-CoA synthetase where an inorganic phosphate attaches and removes CoA. Pi acts as a nucleophile that attaches to a carbonyl and recycles CoA. The enzyme-bound succinyl phosphate releases succinate and leaves a histidine residue with Pi attached in the enzyme. This then phosphorylates GDP to produce GTP

Describe the mechanism of succinyl-CoA synthetase

Succinate dehydrogenase

- Flavoprotein covalently bound to enzyme and prevents dissociation

- This is an integral membrane protein

This step of the TCA cycle occurs when succinate is oxidized to fumarate, allowing a hydride transfer to form FADH2 from FAD. This reaction is near equilibrium and is reversible, so Keq = 1 and DG = 0. The product concentration is kept low to pull the reaction forward. This enzyme is bound to the mitochondrial inner membrane and acts as complex II in the ETC. The reduction of the alkane to alkene here requires FADH2

Ubiquinone

FADH2 needs to be oxidized, so ____________ in the membrane oxidizes FADH2 to FAD to release electrons that are used to produce ATP

Fumarase

In this step of the TCA cycle, water is added across the double bond of fumarate to form malate. This is a stereospecific reaction, so the addition of water is always trans and forms L0malate. OH- adds to fumarate and then H+ is added to the carbanion transition state. This is slightly thermodynamically favorable /reversible, and product concentration is kept low to pull the reaction forward

L-malate dehydrogenase

- Oxaloacetate concentration kept very low by citrate synthase to pull the reaction forward

- Malate concentration kept high

This is the final reaction in the TCA cycle which converts malate to oxaloacetate and completes the reduction of NAD+ to NADH. In standard state conditions, this reaction is highly thermodynamically unfavorable/reversible, but the actual free energy is different because it uses the actual concentrations of malate and oxaloacetate. Overall, the increase in malate concentration and lowering of oxaloacetate concentration makes the reaction favorable in vivo. This regenerates oxaloacetate for citrate synthase

Isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex, and malate dehydrogenase

Which steps from the TCA cycle produce NADH?

Succinyl-CoA synthetase

Which step from the TCA cycle produce GTP which eventually becomes ATP?

Succinate dehydrogenase

Which step from the TCA cycle produces FADH2?

Isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase complex

Which two steps from the TCA cycle release carbon dioxide?

Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O → 2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+

What is the net equation of the citric acid cycle?

Net oxidation of two carbons to CO2 (equivalent to two carbons of acetyl-CoA, but not the same carbons), energy captured by electron transfer to NADH and FADH2, generation of 1 GTP which can be converted to ATP, and completion of the cycle

What are the net results of the TCA cycle?

2.5 ATP

How many ATP are generated from oxidative phosphorylation using 1 NADH?

1.5 ATP

How many ATP are generated from oxidative phosphorylation using 1 FADH2?

Amphibolic

TCA cycle intermediates are ___________, which means they are both catabolic (breaking down) and anabolic (building up). There is a pool of intermediates that are broken down and used to produce energy or other products

Anaplerotic enzymes

These enzymes replenish TCA cycle intermediates by completing side reactions. Intermediates in the citric acid cycle can be used in biosynthetic pathways (removed from the cycle), so they must be replenished in order for the cycle and central metabolic pathway to continue

Pyruvate carboxylase

This anaplerotic reaction occurs in the liver and kidneys to regenerate oxaloacetate + ADP + Pi from pyruvate + bicarbonate + ATP

PEP carboxykinase

This anaplerotic reaction occurs in the heart and skeletal muscle to regenerate oxaloacetate + GTP from PEP + CO2 + GDP

PEP carboxylase

This anaplerotic reaction occurs higher in plants and in yeast and bacteria to regenerate oxaloacetate + Pi from PEP and bicarbonate

Malic enzyme

This anaplerotic reaction is widely distributed in eukaryotes and bacteria to regenerate malate + NAD(P)+ from pyruvate + bicarbonate + NAD(P)H

Biotin

This is a cofactor that exists in the pyruvate carboxylase reaction and acts as a CO2 carrier. The production of oxaloacetate from pyruvate uses this cofactor which is covalently bound through a lysine residue. This transfers CO2 from one active site to another

Dihydrolipoyl transacetylase (E2)

This part of the pyruvate dehydrogenase complex catalyzes the transfer of an acetyl group to coenzyme A.

Fatty acid

Acyl carrier proteins are important biological tethers for __________ ________ biosynthesis

Biological tethers

These components are all covalently linked to residues of enzymes and are substrate-channeling mechanisms. tHese have active groups that do the chemical work. Some examples are dihydrolipoyl transacetylase (E2), pyruvate carboxylase, and acyl carrier protein

Negative: ATP, acetyl-CoA, NADH, fatty acids

Positive: Substrates and negative regulators (products) - AMP, CoA, NAD+, Ca2+

- Positive general regulator = activated by substrate availability

- Negative general regulator = inhibited by product accumulation

What are the positive and negative regulators for the pyruvate dehydrogenase complex?

Pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase

- These first four enzymes are favorable and all have large, negative DG values

What are the main regulators of the TCA cycle?

Ca2+

This ion is a positive regulator for pyruvate dehydrogenase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase

AT highly thermodynamically favorable and irreversible steps = PDH, citrate synthase, IDH, and KDH

Where is the citric acid cycle regulated at?

Inhibitors: NADH and ATP

Activators: NAD+ and AMP

The overall products of the TCA cycle are NADH and ATP, so they affect all regulated enzymes in the cycle. What are the two inhibitors and the two activators that correspond to them?

Allosteric effectors

These molecules are not necessarily the substrate of the reaction, but they enter the pathway through alternative binding sites and affect the active sites

PDH kinase phosphorylates E1 to inactivate it and PDH phosphatase dephosphorylates E1 to activate it. PDH kinase and PDH phosphatase are part of the mammalian PDH complex, and kinase is activated by ATP (high ATP = phosphorylated PDH = less acetyl-CoA ; low ATP = less active kinase and phosphatase removes phosphate from PDH = more acetyl-CoA)

Describe the regulation of pyruvate dehydrogenase complex (PDH) through reversible phosphorylation of E1

Succinyl-CoA

Citrate synthase is inhibited by __________-___________. Alpha-ketoglutarate is an important branch point for amino acid metabolism, and ___________-__________ communicates flow at this branch point to the start of the cycle

Note: Alpha-ketoglutarate dehydrogenase does not have a kinase and a phosphatase, but citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase are similar to the PDH complex reaction

- Organisms have multiple ways to replenish intermediates that are used in other pathways

- The rules of organic chemistry help to rationalize reactions in the citric acid cycle

- The CAC is largely regulated by availability of substrates and product inhibition (especially NADH and ATP)

Chapter 16 Summary (both cards):

- A large multi-subunit enzyme, pyruvate dehydrogenase complex, converts pyruvate into acetyl-CoA

- Several cofactors are involved in reactions that harness the energy from pyruvate

- The CAC is an important catabolic process; it makes GTP and reduced cofactors that could yield ATP

- The CAC plays important anabolic roles in cells