14 - the Citric Acid Cycle

enzymes and amounts of them in pyruvate dehydrogenase

multiple copies of E1, E2, E3

where are E1, E2, and E3 located in eukaryotes

the mitochondria

what does pyruvate dehydrogenase reaction look like

in some bacteria, the 4600-kD pyruvate dehydrogenase complex consists of a cubic core of ____, which are surrounded by ____

24 E2 subunits; an outer shell of 24 E1 and 12 E3 subunits

In mammals and some other bacteria, the enzyme complex is even ___, with ____

larger; 42–48 E1, 60 E2, and 6–12 E3 plus additional

proteins

5 steps of operation of the pyruvate dehydrogenase complex

1. E1 decarboxylates pyruvate; CO2 is released.

2. A leftover hydroxyethyl group is transferred to E2.

3. An acetyl group is transferred to CoA.

4. E2 is restored.

5. E3 is restored and NADH is produced.

Step 1 AKA pyruvate dehydrogenase does what? requires what?

catalyzes pyruvate, which is decarboxylated

requires the cofactor thiamine pyrophosphate (TPP)

what is thiamin

vitamin B1

pyruvate dehydrogenase reaction step 2 does what

The hydroxyethyl group is then transferred to E2 of the pyruvate dehydrogenase complex.

The transfer reaction regenerates the TPP cofactor of E1 and oxidizes the hydroxyethyl group to an acetyl group

active portion of step 2

disulfide bond

pyruvate dehydrogenase reaction step 3 does what

E2 transfers the acetyl group to coenzyme A, producing acetyl-CoA and leaving a reduced lipoamide group

acetyl coA is___

a thioester, a form of energy currency

pyruvate dehydrogenase reaction step 4 what happens

E3 reoxidizes the lipoamide group of E2 by transferring electrons to a Cys–Cys disulfide group in the enzyme

pyruvate dehydrogenase reaction step 5 what happens

NAD+ reoxidizes the reduced cysteine sulfhydryl groups. This electron-transfer reaction is facilitated by an FAD prosthetic group.

in step 4 and 5, what happens to the pyruvate dehydrogenase complex?

restored to its original state

During the five-step reaction, the ____ acts as ______ within the multienzyme complex

the long lipoamide group of E2; a swinging arm that visits the active sites of E1, E2, and E3

most important aspects of pyruvate dehydrogenase

enzyme, cofactor, substrate, product, free energy

2 main controls of pyruvate dehydrogenase reaction

feedback inhibitors (NADH, acetyl-CoA)

hormones (phosphorylation/dephosphorylation)

starting material for citric acid cycle; what happens when its carbons become fully oxidized to CO2?

acetyl-CoA (may be derived from carb via pyruvate)

their energy is conserved and subsequently used to produce ATP

The eight reactions of the citric acid cycle take place in the ____ of prokaryotes and in the ____ of eukaryotes

the cytosol of prokaryotes and in the mitochondria of eukaryotes

how does the citric acid cycle compare to linear pathways?

the citric acid cycle always returns to its starting position, essentially behaving as a multistep catalyst.

citric acid cycle reaction 1

Citrate synthase adds an acetyl group to oxaloacetate

citric acid cycle reaction 1 starting/ending materials, enzyme

oxaloacetate + Acetyl-Coa → citrate

citrate synthase

Citrate synthase is one of the few enzymes that can _____

synthesize a carbon–carbon bond without using a metal ion cofactor

citrate synthase acts as a dimer that undergoes a _____ upon binding oxaloacetate, allowing for binding of acetyl-CoA

conformational change

free energy change of reaction catalyzed by citrate synthase

highly exergonic; deltaG = -31.5 kJ/mol

citric acid cycle reaction 2

Aconitase isomerizes citrate to isocitrate

citric acid cycle reaction 2 substrates/products

citrate → intermed aconitase → isocitrate

Citrate is a symmetrical molecule, but binding to an asymmetrical enzyme leads to ___

stereochemical specificity

citric acid cycle reaction 3

isocitrate dehydrogenase releases the first CO2

citric acid cycle reaction 3 substrate/product

isocitrate → alpha-keoglutarate

citric acid cycle step 4

α-Ketoglutarate dehydrogenase releases the second CO2

citric acid cycle step 4 substrate/enzyme

α-Ketoglutarate → succinyl-CoA

α-Ketoglutarate dehydrogenase is a multienzyme complex that resembles _____

the pyruvate dehydrogenase

citric acid cycle step 5

Succinyl-CoA synthetase catalyzes substrate-level phosphorylation

Succinyl-CoA synthetase in the mammalian citric acid cycle generates ____ whereas the plant and bacterial enzymes generate ____

GTP; ATP

in citric acid cycle reaction 5, the thioester succinyl-CoA releases a ____ amount of free energy when it is hydrolyzed

large

citric acid cycle reaction 5 exergonic reaction coupled to the transfer of a phosphoryl group to a nucleoside diphosphate is termed ___

substrate-level phosphorylation

citric acid cycle reaction 6

succinate dehydrogenase generates ubiquinol

citric acid cycle reaciton 6 substrate/products/enzyme

succinate → fumarate

succinate dehydrogenase

Succinate dehydrogenase catalyzes the reversible dehydrogenation of succinate to fumarate. This oxidation-reduction reaction requires an _____, which is reduced to ____ during the reaction.

FAD prosthetic group; FADH2

citric acid cycle step 7

fumarase catalyzes a hydration reaction

citric acid cycle step 7 substrate/product/enzyme

fumarate → malate

fumarase

citric acid cycle reaction 8

Malate dehydrogenase regenerates oxaloacetate

citric acid cycle reaction 8 substrate/product/enzyme

malate → oxaloacetate

malate dehydrogenase

reaction 8 has a high free energy change/low likelihood of occuring, but oxaloacetate is substrate for the next reaction (1), so the highly exergonic (favorable) citrate synthase reaction helps pull the malate dehydrogenase reaction forward, which the reason for?

the apparent waste(or investment) of free energy released by cleaving the thioester bond of acetyl-CoA in the first reaction of the citric acid cycle.

every acetyl unit that enters the citric acid cycle can generate a total of___

10 ATP equivalents

Because the eighth reaction of the citric acid cycle returns the system to its original state, the entire pathway acts in a ____ fashion to dispose of _____ derived from ____

catalytic fashion

carbon atoms

amino acids, carbohydrates, fatty acids

A muscle operating anaerobically produces ___ per glucose; under aerobic conditions, it produces ___

only 2 ATP; 32 ATP equivalents

Pasteur effect

A muscle operating anaerobically produces only 2 ATP per glucose, but under aerobic conditions when the citric acid cycle is fully functional, each glucose molecule generates about 32 ATP equivalents.

which three reactions is the citric acid cycle regulated at?

the 3 metabolically irreversible steps: citrate synthase (1), isocitrate dehydrogenase (3), alpha-ketoglutarate dehydrogenase (4)

citric acid cycle reaction 1 activators, inhibitors

activators: ADP

inhibitors: NADH, succincyl-CoA, citrate

citric acid cycle reaction 3 activators, inhibitors

activators: ADP and Ca2+

inhibitors: NADH, ATP

citric acid cycle reaction 4 activators, inhibitors

activators: Ca2+, AMP

inhibitors: NADH, succinyl-CoA

In mammals, ___of the eight citric acid cycle intermediates (all except ______) are the precursors or products of other pathways.

6

isocitrate, succinate

makes it impossible to designate as purely catabolic or anabolic

succinyl-CoA is used for the synthesis of

heme

alpha-ketoglutarate can undergo reductive amination by ___ to produce___

glutamate dehydrogenase; amino acid glutamate

glutamate is a precursor of the amino acids__

glutamine, arginine, proline

glutamine is a precursor for the synthesis of ___

purine and pyrimidine nucleotides

oxaloacetate is a precursor of

monosaccharides

citrate can be transported out of the mito to the cytosol, undergo a reaction catalyzed by ____ to produce ____, used for ___

ATP-citrate lyase

acetyl CoA

fatty acid and cholesterol synthesis

oxaloacetate can be converted to ___, which is then decarboxylated to produce ____

malate

pyruvate

why is cytosolic ATP-citrate lyase essential?

because acetyl-CoA, which is produced in the mitochondria, cannot cross the mitochondrial membrane to reach the cytosol, whereas citrate can.

In plants, isocitrate is diverted from the citric acid cycle in a biosynthetic pathway known as the

glyoxylate pathway, named after an organelle, glyoxysome in plant cells

the glyoxylate pathway provides a route for

synthesizing glucose from fatty acids

because animals lack _____, they cannot convert excess ___ to ___

lyase and malate synthase

fat to carbohydrate

Intermediates that are diverted from the citric acid cycle for other purposes can be replenished through

anaplerotic reactions.

what does the citric acid cycle act as? how does this affect flux through the pathway?

acts as a catalyst

increasing the concentrations of its components increases flux through the pathway

Degradation of fatty acids with an odd number of carbon atoms yields

succinyl-CoA

Degradation of some amino acids produces

alpha-ketoglutarate, succinyl-CoA, fumarate, and oxaloacetate.

how does concentration of citric acid cycle intermediates change in vigorously exercising muscle?

increases about 3-4x fold

how does concentration of citric acid cycleflux change in vigorously exercising muscle?

up to 100x fold

why how does concentration of citric acid cycle flux increase so greatly in vigorously exercising muscle?

As a result from rapid glycolysis at the start of exercise. Not all of this pyruvate is converted to lactate; some is shunted into the pool of citric acid cycle intermediates via the pyruvate carboxylase reaction. Some pyruvate also undergoes a reversible reaction catalyzed by alanine aminotransferase: they are converted to α-ketoglutarate to boost the activity of the citric acid cycle. The resulting α-ketoglutarate then augments the pool of citric acid cycle intermediates, thereby increasing the ability of the cycle to oxidize the extra pyruvate