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Home , Aconitase, Arsenite, Catabolism, Citric acid, Citric acid cycle, Coenzyme A

Overview of the citric cycle, AKA the krebs cycle AKA tricarboxylic acid AKA TCA cycle Suggested problems from the end of chapter 19: 1,2,3,5,6,8,10,11,16,20,21

Glycogen is broken down into . The reactions of result in pyruvate, which is then fed into the cycle in the form of acetyl CoA.

The products of the citric acid cycles are 2 CO2, 3 NADH, 1 FADH2, and 1 ATP or GTP.

After pyruvate is generated, it is transported into the , an that contains the and the oxidative enzymes. In E. coli, where there are neither mitochondria nor other , these enzymes also seem to be concentrated in certain regions in the .

Citric acid cycle reactions

Overall, there are 8 reactions that result in oxidation of the metabolic fuel. This results in reduction of NAD+ and FAD.

NADH and FADH2 will transfer their to during oxidative phosphorylation.

•In 1936 Carl Martius and Franz Knoop showed that citrate can be formed non-enzymaticly from Oxaloacetate and pyruvate. •In 1937 used this information for biochemical experiments that resulted in his suggestion that citrate is processed in an ongoing circle, into which pyruvate is “fed.” •In 1951 it was shown that it was acetyl Coenzyme-A that condenses with oxaloacetate to form citrate.

1 The pre-citric acid reaction- pyruvate

Pyruvate dehydrogenase is a multi-subunit complex, containing three enzymes that associate non-covalently and catalyze 5 reaction. The enzymes are:

(E1) (E2) dihydrolipoyl transacetylase (E3) dihydrolipoyl dehydrogenase

What are the advantages for arranging enzymes in complexes?

E. coli pyruvate dehydrogenase complex

The E. coli complex is a particle of about 300 Å in diameter. The mass is ~4.6 million Daltons (g/mol). There are 60 polypeptides per particle.

The complex contains 24 E2 which are arranged in a cube and form the so-called core complex. 24 E1 proteins and 12 E3 proteins are arranged around the core.

2 The E. coli and pyruvate dehydrogenase complexes share structural features

“Core” complex, E2 (dihydrolipoyl transacetylase) proteins associate as trimers at the corners of the cube.

E1 (pyruvate dehydrogenase) dimers (in yellow/) associate with the

E2 trimers at the edges of the cube.

E3 (dihydrolipoyl dehydrogenase) dimers (light blue) associate at the six faces of the cube.

Pyruvate dehydrogenase catalyzes five reactions

3 Pyruvate dehydrogenase reactions Reaction 1 involves of pyruvate in a reaction that depends on the co- (TPP). The mechanism resembles that of the

reaction, except the product here is hydroxyethyl TPP:E1.

Thiamine pyrophosphate

H3C O O O O Aminopyrimidine -O C O H C C O P O P O- -C ring 2 H H O C O + C N 2 2 C N - S O O- R Thiazolium R ring H H3C N NH2

H3C Hydroxyethyl-TPP:E1

R' R N+ S

- H O C CH3

Pyruvate dehydrogenase reactions

In reaction 2 the hydroxyethyl group is transferred to dihydrolipoyl transacetylase (E2). The E2 co-enzyme is a lipoamide group. The lipoamide is attached to the enzyme via an linkage to a side chain. The lipoamide can interconvert with the dihydrolipoamide form.

4 Pyruvate dehydrogenase reactions In reaction 2 of pyruvate dehydrogenase the hydroxyethyl group attacks the lipoamide disulfide. The hydroxyethyl is oxidized to an and the lipoamide group is reduced.

Pyruvate dehydrogenase reactions

Reaction 3. E2 catalyzes a transesterification reaction, in which the acetyl group is transferred to CoA. Acetyl-CoA will be used in the citric acid cycle, but the dihydrolipoamide group must

be oxidized to regenerate the E2 enzyme.

5 Thioesters are considered “primitive” high- compounds. They are found in the central metabolic pathways of all known . Thioesters were, presumably, more common in the pre- biotic world than compounds. are less abundant in non-living systems than thioesters.

In metabolic pathways the appears as a part of acetyl- (CoA). This is an intermediate in , , and .

∆Gº’ of acetyl-CoA is -31.5 kJ/mol.

Pyruvate dehydrogenase reactions

Reaction 4. E3, dihydrolipoyl dehydrogenase, is used to regenerate the lipoamide group. A reactive cys-cys disulfide group is used together with a non-covalent, tightly bound FAD.

Reaction 5. Re-generation of E3. The sulfhydryl groups are oxidized by FAD, and FAD is oxidized by NAD+, which results in the production of NADH.

6 Pyruvate dehydrogenase reactions

The E2 on the inside of the structure must “channel” intermediates from the E1 and E3 enzymes on the outside of the structure. This apparently is achieved due to the flexibility and length of the lipoyllysyl arm.

This arm (shown on right) may swing between E1 and E3. In terms of the reactions that are catalyzed, this arm can be thought of as a “tether” that brings the reactive disulfide group to the of E1 (where it reacts to accept the hydroxyethyl group), then swings to the active site of E2 (where CoA accepts the acetyl group), and finally to the active site of E3 (where the disulfide group is re-oxidized).

Support for this idea is the finding that the lipoyllysyl arm is attached to the E2 protein via an - rich region that seems to adopt multiple conformations, meaning that it is flexible. If this idea is correct, a single lipoyllysyl arm can reach numerous E3 active sites.

How would you test the idea that a flexible linkage near the lipoamide group is essential for the activity of pyruvate dehydrogenase?

Structure of E2 of pyruvate dehydrogenase

The E2 catalytic domain from the bacterium vinelandii is shown.

The 24 E2 proteins are arranged in trimers, each trimer occupying the corner of a cube. The edge of the cube is ~125 Å. Substrates and co-factors must

fit in the spaces between the E2 proteins and within the interior cavity of the cube. Only 4 “corners” are shown.

Acetyl CoA must diffuse into its from the internal space of the cube.

7 effects on pyruvate dehydrogenase

S OH HS

-O As + -O As + 2 H2O

OH HS S

R R Arserite Dihydropiloamide

3- • compounds (AsO3 ) and organic arsenicals (R—As=O) inhibit aerobic respiration by reacting with lipoamide-containing enzymes (pyruvate dehydrogenase is just one of these enzymes). Asrenicals also can affect bacterial enzymes, and apparently at lower doses, which is the reason for the treatment of syphilis and trypanosomiasis with arsenicals before the advent of modern antibiotics. Side effects were severe. •Arsenic may be consumed unwittingly. Napoleon, for example, may have suffered a chronic poisoning. Many wallpaper dyes at that time contained arsenic, and growing on the walls in damp weather (for example, on Elba) can convert the arsenic to volatile form, which can then be ingested. •Fowler ’s tonic, which many people took to remedy various ailments, contained 10 mg/ml arsenite.

Reaction 1. Citric - proposed catalytic mechanism The reaction involves condensation of acetyl CoA and oxaloacetate into citric acid. His-274, -320, and aspartate-375 have been implicated in general acid - general base .

8 Reaction 1. Citric Synthase - proposed binding mechanism

oxaloacetate

There is no binding affinity detected for acetyl-CoA in the absence of oxaloacetate. Oxaloacetate is bound in the presence or absence of acetyl-CoA. In the presence of oxaloacetate alone there is no catalytic reaction, but the above conformational change is observed. What substrate binding mechanism is supported by these observations?

Reaction 2.

The enzyme contains a 4Fe-4S - cluster. The enzyme is unusual in that it does not catalyze a reaction, as most iron-sulfur enzymes do. In the second stage of this reaction, addition of across the the double bond can, in principle, result in 4 stereoisomers. The enzyme is stereospecific in the sense that only one isocitrate stereoisomer is produced by the enzyme.

9 Reaction 2. Aconitase

The iron not bound to a sulfur coordinates the C3 OH and COO- groups of citrate. This iron accepts an from the hydroxyl group, which then becomes the leaving group.

Reaction 3.

+ This reaction produces the first CO2 and NADH of the citric acid cycle. The enzyme is NAD - dependent

•To achieve the decarboxylation reaction, magnesium or manganese can be utilized by the enzyme.

•The CO2 that is released originated from oxaloacetate (not acetyl-CoA). •Specific in this enzyme help elucidate the catalytic mechanism because these mutations did not completely destroy catalysis, rather these mutations slowed some of the steps in the pathway such that intermediates accumulated. For example, the presence of oxalosuccinate was not observed until mutations in lysine-230 and -160 were made. These mutations act as kinetic “bottlenecks” such that reactions involving the intermediates are slowed down.

10 Reaction 4. α-ketoglutarate dehydrogenase

This reaction resembles the pyruvate dehydrogenase multi-enzyme complex reaction, down to the identity of the E3 dihydrolipoyl dehydrogenase. As expected, the mechanisms are, most likely, the same, and the product is the high energy thioester, in this case, succinyl CoA.

Reaction 5. Succinyl CoA synthetase

Succinyl-CoA + ADP + Pi ↔ ATP + succinate + CoASH

This enzyme couples the free energy released from hydrolysis of the high energy thioester succinyl CoA to the synthesis of a high energy NTP. GTP is generated in mammalian cells, whereas ATP is generated in and plant cells. From an energetic standpoint, these reactions essentially are the same because GTP and ATP can be interconverted rapidly through the action of nucleoside diphosphate .

GTP + ADP ↔ GDP + ATP ∆G°’ = 0

Isotopic labeling experiments permit us to “follow” the fate of the labeled group and elucidate the catalytic steps. In the absence of succinyl CoA, the enzyme transfers the unlabeled γ-phosphoryl group of ATP to a labeled ADP, thus generating labeled ATP. What substrate binding mechanism is supported by this finding?

11 Reaction 5. Succinyl CoA synthetase

Because the enzyme can catalyze the exchange reaction, the existence of an enzyme-intermediate complex was proposed. This complex, termed E’ in the general scheme of the ping-pong mechanism, is often a between the enzyme and the intermediate. A kinetically active (albeit slowed down) mutant of the enzyme was found, in which a histidine N3 was covalently linked to a phosphoryl group.

Reaction 5. Proposed catalytic mechanism of succinyl CoA synthetase

12 Reaction 6.

The enzyme catalyzes the oxidation of succinate to fumarate, with the concomitant reduction of FAD to FADH2. The enzyme is strongly inhibited by malonate, which is a classic example of a structural analog exhibiting .

-OOC C COO- H2 malonate

Reaction 6. Succinate dehydrogenase

This enzyme is unusual in that the FAD group is covalently attached, whereas in most other FAD- containing enzyme FAD is bound tightly but non-covalently. Here the FAD group is attached to the N3 of histidine.

E

CH2

histidine N R FAD N C N N O H2

N

H3C N H

O

Unlike the rest of the citric acid cycle enzymes, succinate dehydrogenase is bound within the mitochondrial membrane. The other enzymes are in the .

13 Reaction 7.

The double bond of fumarate is hydrated to form malate. The enzyme yields only one stereoisomer.

Reaction 8.

NADH is generated in the 8th and last step of the cycle. The NAD+ binding domain of malate dehydrogenase, , and dehydrogenase all look remarkably similar. In terms of binding site , what can you conclude?

14 Regulation of the citric acid cycle - is there a ?

According to the metabolon hypothesis, enzymes involved in a pathway might be expected to be physically associated in vivo. The same considerations given for the efficiency of pyruvate dehydrogenase can be used here successfully. At least two criteria must be met:

1. In vitro evidence for the association of proteins must pass the test of specificity. Many proteins can associate in vitro, but what is the relevance of such data to the situation in vivo? Specificity should be demonstrated. By one criterion, specificity is measured by the resistance to non-specific competition.

2. Evidence in vivo must be obtained. This can be done through (fluorescent) labeling that can demonstrate co-localization of the proteins in the cell. Also, this can be done by isolating sub-organelle structures containing the pathway enzymes. At least some of the citric acid cycle proteins seem to associate (transiently or weakly) with the inner mitochondrial membrane. Partially purified mitochondrial fractions display a faster turnover rate than completely solubilize fractions, arguing that there is an advantage to keeping the enzymes in their “in vivo” overall arrangement.

Regulation of pyruvate dehydrogenase

The entry of acetyl-CoA into the citric acid cycle results in a large-scale production of ATP through oxidative phosphorylation. It is not surprising that the citric acid cycle is regulated carefully, especially because there is no other enzyme that will generate acetyl CoA from pyruvate in mammalian cells. The first reaction, the charging of CoA with an acetyl group, via the action of the multi-enzyme complex pyruvate dehydrogenase, is not reversible (operates far form equilibrium). This is a natural site for enzymatic regulation.

1. Product inhibition by NADH and acetyl CoA. These products compete with NAD and CoA, respectively, for their binding sites. High [NADH]/[NAD+] and [acetyl-CoA]/[CoA] result in

enzyme inhibition. E1 is the non-reversible enzyme within this complex.

2. The E1 (pyruvate dehydrogenase) activity is modified by phosphorylation and de-phosphorylation, such that when the enzyme is phosphorylated it is inactive.

15 Regulation of pyruvate dehydrogenase (continued)

Insulin is released in response to high glucose levels. activates pyruvate dehydrogenase . This results in the de-phosphorylation of pyruvate dehydrogenase and activation of the citric acid cycle. In addition to activating the glucose metabolic pathways, insulin stimulates the anabolic pathways, thus helping to maintain steady levels of blood glucose.

3. Pyruvate and ADP inhibit pyruvate dehydrogenase kinase.

Rate-controlling enzymes of the citric acid cycle

The difficulty in defining the ∆G of the citric acid reactions is that many of the are present in the and the mitochondria, but the concentrations of these metabolites in each compartment are not known. Under the assumption that metabolites are free to diffuse into the mitochondria, ∆G can be calculated. Only reactions 1, 3, and 4 show ∆G < 0, which argues that these reactions are the rate limiting steps in the pathway.

The citric acid enzymes are controlled via three mechanisms: 1. Substrate availability. 2. Product inhibition. 3. Competitive feedback inhibition by intermediates. Thus most of the regulation depends on the amount of substrates being fed into the cycle (acetyl-CoA) and the cycle products (ATP, NADH).

16 Regulation of citric acid cycle reactions

Citric acid intermediates are used in other pathways 1. Oxaloacetate can be used for .

2. Amino acid utilizes α-ketoglutarate in a reaction catalyzed by .

COO- COO-

CH2 CH2

CH + + CH + NAD+ + H O 2 + NADH + H + NH4 2 2

+ C O H C NH3

COO- COO-

α-Ketoglutarate Glutamate

17 Citric acid intermediates are used in other pathways

3. Aspartate aminotransferase catalyzes the trans-amination reaction that yields aspartate from oxaloacetate.

COO- COO- COO- COO- + C O H3N C H + + H3N C H + C O

CH2 CH2 CH3 CH3 COO- COO-

Oxaloacetate Alanine Aspartate Pyruvate

18