Ch. 16: The Citric Acid Cycle

What are two other names for the citric acid cycle?

tricarboxylic acid cycle (TAC)

Krebs cycle

Compare the energy released from glycolysis to the energy released from complete oxidation of the products?

glycolysis is favorable and releases a good amount of energy

complete oxidation to CO2 and H2O produces almost 20x the amount of energy

Cellular respiration is the process in which cells consume ____ and produce ____.

O2

CO2

Cellular respiration provides more energy (ATP) from glucose than _______ and also captures energy stored in ___ and ___.

glycolysis

lipids and amino acids

Three major stages of cellular respiration?

1. acetyl CoA production

2. acetyl CoA oxidation

3. electron transfer and oxidative phosphorylation

Stage 1 of cellular respiration (acetyl-CoA production) generates _____ of the total potential CO2 from carbohydrates.

1/3

Stage 2 of cellular respiration (acetyl-CoA oxidation) generates ___, ____, and one _____.

NADH

FADH2

GTP

Stage 2 of cellular respiration (acetyl-CoA oxidation) releases ___ of the carbon atoms from carbohydrates.

2/3

In Stage 2 of cellular respiration (acetyl-CoA oxidation), acetyl-CoA is converted into ____.

citrate

Stage 3 of cellular respiration (oxidative phosphorylation) requires _____ and generates ___ and the vast majority of ___ during catabolism.

oxygen

water

ATP

Where do glycolysis, Kreb's cycle, and oxidative phosphorylation take place in cell?

glycolysis: cytoplasm

CAC: mitochondrial matrix

Oxidative ph.: inner membrane

All parts of the citric acid cycle except _____ occur in the mitochondrial matrix. This occurs in the inner membrane.

succinate dehydrogenase

What is the reaction between glycolysis and the citric acid cycle?

conversion of pyruvate to acetyl Co-A

The conversion of pyruvate to acetyl CoA is catalyzed by the ____.

pyruvate dehydrogenase complex

In the conversion of pyruvate to acetyl-CoA, what are the three prosthetic groups?

TTP

Lipoyllysine

FAD

In the conversion of pyruvate to acetyl-CoA, what are the two co-substrates?

NAD+

CoA-SH

What is the first reaction in cellular respiration that produces CO2?

conversion of pyruvate to acetyl-CoA

The conversion of pyruvate to acetyl-CoA is (reversible/irreversible).

irreversible

Are coenzymes a permanent part of an enzyme's structure?

no

What is the function of CoA?

to accept and carry acetyl groups

What is the active part of Coenzyme A?

thiol group

What is the structure of lipoyllysine?

lipoic acid covalently linked (with amide linkage) to enzyme via lysine residue

Three forms of lipoyllysine?

oxidized form

reduced form

acetylated form

Pyruvate dehydrogenase complex (PDC) is a large (up to ___ MDa) ______ complex.

10

multienzyme

What are E1-E3 of the pyruvate dehydrogenase complex?

E1 = pyruvate dehydrogenase

E2 = dihydrolipoyl transacetylase

E3 = dihydrolipoyl dehydrogenase

What are the advantages of multienzyme complexes?

1) Increased rate due to decreased distances between active sites

2) Minimizes chances for side reactions by channeling the product of each reaction to the next active site

3) Coordinate regulation of the reactions

What is a good method to find the structure of large protein complexes such as PDC?

cryoelectronmicroscopy

What steps occur at enzyme one for oxidative decarboxylation of pyruvate?

Step 1: oxidative decarboxylation of pyruvate, bound to TPP in hydroxyl ethyl intermediate (CO2 formed)

Step 2: oxidation of hydroxyl ethyl to a carbonyl; electrons (hydride) reduce lipoamide and form a thioester

What steps occur at enzyme two for oxidative decarboxylation of pyruvate?

Step 3: formation of acetyl-CoA (product two)

What steps occur at enzyme three for oxidative decarboxylation of pyruvate?

Step 4: reoxidation of the lipoamide cofactor

Step 5: regeneration of the oxidized FAD cofactor; formation of NADH (product 3)

Sequence of events in the citric acid cycle:

Step 1: ______ bond formation between ____ and ___ to make _____

Step 2: Isomerization via ______/________

Step 3-4: Oxidative decarboxylations to give _____________

Step 5: _____________ to give GTP

Step 6: ______ to give _____

Step 7: _____

Step 8: ______ to give ____

1: C-C, acetate, oxaloacetate, citrate

2: dehydration/rehydration

3: 2 NADH

5: substrate-level phosphorylation

6: dehydrogenation, FADH2

7: hydration

8: dehydrogenation; NADH

Citrate synthesis is a ____ type of reaction.

condensation

______ and _____ merge carbons to form citrate.

acetyl-CoA and oxaloacetate

What is the only reaction with C-C bond formation?

citrate synthase

What type of catalysis does citrate synthase use?

acid/base

What is the rate-limiting step of the citric acid cycle?

citrate synthase

Citrate synthase has activity dependent on the concentration of _____.

oxaloacetate

How favorable is citrate synthase?

highly thermodynamically favorable / irreversible

The conformation change that occurs when oxaloacetate binds in citrate synthase is an example of ____.

induced fit

What is the open conformation of citrate synthase?

free enzyme that does not have a binding site for acetyl-CoA

What is the closed conformation of citrate synthase?

binding of oxaloacetate creates binding for acetyl-CoA

The binding of oxaloacetate increases what in citrate synthesis?

the affinity for acetyl-CoA binding

________ is a common amino acid found at active sites that use acid-base catalysis.

histadine

In citrate synthase in the mechanism of hydrolysis of thioester, how is Co-A gotten rid of?

hydrolysis reaction

After citrate is produced as a product, it goes on to be what?

a substrate for the aconitase reaction

Describe the isomerization by dehydration/rehydration of citrate into isocitrate.

Citrate loses hydroxyl group and H to produce double bond (cis-aconitate) then gains water back on to form new hydroxyl group and hydrogen = isocitrate

Citrate is a _____ alcohol and a ____ substrate for oxidation.

tertiary

poor

Isocitrate is a ___ alcohol and a ___ substrate for oxidation.

secondary

good

Is the aconitase reach favorable or unfavorable?

thermodynamically unfavorable / reversible

Iron-Sulfur Center in Aconitase

Water removal from citrate and subsequent addition to cis-aconitate are catalyzed by the iron-sulfur center: sensitive to oxidative stress.

Isozymes are specific for ____ (where?) and ___ (where?).

NADP+ (cytosolic)

NAD+ (mitochondrial)

Isozymes

Different enzymes that catalyze the same reaction

Typically share similar sequences

May have different kinetic properties

Can be regulated differently

In oxidative decarboxylation by isocitrate dehydrogenase, what is released and what is reduced?

CO2

NAD+ reduced to NADH

How favorable is the oxidative decarboxylation by isocitrate dehydrogenase?

highly favorable / irreversible

What regulates oxidative decarboxylation by isocitrate dehydrogenase?

[ATP]

What enzyme is involved in the final oxidative decarboxylation in CAC?

a-ketoglutarate dehydrogenase complex

The a-ketoglutarate dehydrogenase complex is very similar to what?

the pyruvate dehydrogenase complex

How is the a-ketoglutarate dehydrogenase complex similar to the pyruvate dehydrogenase complex? Different?

Same: same coenzymes with identical mechanisms

Different: active sites different to accommodate different sized substrates (a-ketoglutarate vs. pyruvate)

Net full oxidation of all carbons of glucose occurs after ___ turns of the cycle.

two

The a-ketoglutarate dehydrogenase reaction is how favorable?

highly thermodynamically favorable / irreversible

The a-ketoglutarate dehydrogenase reaction is regulated by what?

product inhibition (NADH and Acetyl-CoA)

All CO2 generated during the citric acid cycle is produced before _____ is made.

succinyl-CoA

Succinyl-CoA becomes Succinate using what enzyme?

succinyl-CoA synthetase

What is produced during the succinyl-CoA synthetase reaction?

GTP

Through what is GTP produced in the succinyl-CoA synthetase?

substrate-level phosphorylation

In the succinyl-CoA synthetase, the energy of the ______ allows for incorporation of inorganic phosphate.

thioester

What enzyme catalyzes ADP + GTP <-> ATP + GDP?

nucleoside-diphosphate kinase

How favorable is the succinyl-CoA synthetase reaction?

slightly thermodynamically favorable / reversible

How is the reaction pulled forward in the slightly favorable succinyl-CoA synthetase reaction?

product concentration kept low

Succinate becomes fumarate using what enzyme?

succinate dehydrogenase

What is unique about succinate dehydrogenase?

it is an integral membrane protein with FAD covalently bound

In the succinate dehydrogenase reaction, what are the two products?

FADH2

Fumarate

What reaction is near equilibrium and therefore has a free energy of 0?

succinate dehydrogenase reaction

What enzyme is used to transform fumarate into L-Malate?

fumarase

How is fumarase stereospecific?

the addition of water is always trans and forms L-Malate

How favorable is the fumarase reaction?

slightly thermodynamically favorable / reversible

What enzyme is used to transform L-Malate into Oxaloacetate?

L-malate dehydrogenase

What are the three products of the L-malate dehydrogenase reaction?

NADH

H+

oxaloacetate

Malate dehydrogenase is the final step of the cycle that regenerates oxaloacetate for __________.

citrate synthesis

How favorable is the malate dehydrogenase reaction?

highly thermodynamically unfavorable / reversible

Because the maltase dehydrogenase reaction is thermodynamically unfavorable, how is the reaction pulled forward?

oxaloacetate concentration kept VERY low by citrate synthase

In the CAC, what three reactions produce NADH?

isocitrate dehydrogenase

a-ketoglutarate dehydrogenase

malate dehydrogenase

In the CAC, CO2 is released during what two reactions?

isocitrate dehydrogenase

a-ketoglutarate dehydrogenase

In the CAC, FADH is produced in which reaction?

succinate dehydrogenase

In the CAC, substrate-level phosphorylation occurs in the ____ reaction.

Succinyl-CoA

The Citric Acid Cycle equation:

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

How much ATP is typically produced by 1 NADH? FADH?

2.5 ATP

1.5 ATP

If you completely oxidize glucose, how much ATP would be produced aerobically?

30-32 ATP

Amphibolic pathway

a metabolic pathway used in both catabolism and anabolism

CAC intermediates are ______.

amphibolic

Give examples of CAC intermediates being amphibolic.

a-ketoglutarate becoming glutamate to produce other amino acids

citrate becoming fatty acids, sterols

succinyl-CoA becoming porphyrins, heme

___________ reactions help produce some of the intermediates in the CAC.

Anaplerotic

Give examples of anaplerotic reactions for the CAC intermediates.

malate produced from pyruvate using malic enzyme

oxaloacetate produced from pyruvate or phosphoenolpyruvate (using pyruvate carboxylase and PEP carboxylase, respectively)

__________ is a CO2 carrier.

Biotin

What does biotin do?

- always performs single carbon transfer

- CO2 is covalently bound to biotin-enzyme and biotin serves as a carrier of activated CO2

- activated CO2 is transferred to pyruvate to form oxaloacetate

Biological _____ allow flexibility.

tethers

What are the three structures we learned that can "swing" carbons from one active site to another?

biotin

lipoate

acyl carrier protein

What 4 things inhibit the pyruvate dehydrogenase complex reaction?

ATP

acetyl-CoA

NADH

fatty acids

What 4 things activate the pyruvate dehydrogenase complex reaction?

AMP, CoA, NAD+, Ca2+

What 4 things inhibit the citrate synthase reaction?

NADH

succinyl-CoA

citrate

ATP