Pyruvate Dehydrogenase & Krebs Cycle

Pathway localization:

Glycolysis enzymes are located in the cytosol of cells.  Pyruvate enters the mitochondrion to be metabolized further. 

Mitochondrial compartments:

           The mitochondrial matrix contains Pyruvate Dehydrogenase and enzymes of Krebs Cycle, plus other pathways such as fatty acid oxidation. The mitochondrial outer membrane contains large VDAC channels, similar to bacterial porin channels, making the outer membrane leaky to ions and small molecules. 

            The inner membrane is the major permeability barrier of the mitochondrion. It contains various transport catalysts, including a carrier protein that allows pyruvate to enter the matrix. It is highly convoluted, with infoldings called cristae. Embedded in the inner membrane are constituents of the respiratory chain and ATP Synthase.

 

 

Pyruvate Dehydrogenase catalyzes oxidative decarboxylation of pyruvate, to form acetyl-CoA. The overall reaction is shown at right.

 

 

Pyruvate Dehydrogenase is a large complex containing many copies of each of three enzymes, E1, E2, and E3. The structure of the complex is as follows:

The inner core of the mammalian Pyruvate Dehydrogenase complex is an icosahedral structure consisting of 60 copies of E2.

At the periphery of the complex are:

  • 30 copies of E1 (itself a tetramer with subunits a2b2) and
  • 12 copies of E3 (a homodimer), plus 12 copies of an E3 binding protein that links E3 to E2.

Prosthetic groups are listed below, a cartoon showing 3 subunits is at right, and a diagram summarizing the reactions catalyzed is on p. 770. 

 

 

Enzyme

Abbreviated

Prosthetic Group

Pyruvate Dehydrogenase

E1

Thiamine pyrophosphate (TPP)

Dihydrolipoyl Transacetylase

E2

Lipoamide

Dihydrolipoyl Dehydrogenase

E3

FAD

FAD (Flavin Adenine Dinucleotide) is a derivative of the B-vitamin riboflavin (dimethylisoalloxazine-ribitol). The flavin ring system undergoes oxidation/reduction as shown below. Whereas NAD+ is a coenzyme that reversibly binds to enzymes, FAD is a prosthetic group, that is permanently part of the complex. 

FAD accepts and donates 2 electrons with 2 protons (2 H):

FAD + 2 e- + 2 H+ ¨ģ FADH2

 

Thiamine pyrophosphate (TPP) is a derivative of  thiamine (vitamin B1). Nutritional deficiency of thiamine leads to the disease beriberi. Beriberi affects especially the brain, because TPP is required for carbohydrate metabolism, and the brain depends on glucose metabolism for energy.

 

A proton readily dissociates from the C that is between N and S in the thiazole ring of TPP. The resulting carbanion (ylid) can attack the electron-deficient keto carbon of  pyruvate.

 

 

Lipoamide includes a dithiol that undergoes oxidation and reduction. 

The carboxyl group at the end of lipoic acid's hydrocarbon chain forms an amide bond to the side-chain amino group of a lysine residue of E2.

A long flexible arm, including hydrocarbon chains of lipoate and the lysine R-group, links the dithiol of each lipoamide to one of two lipoate-binding domains of each E2. Lipoate-binding domains are themselves part of a flexible strand of E2 that extends out from the core of the complex.

The long flexible attachment allows lipoamide functional groups to swing back and forth between E2 active sites in the core of the complex and active sites of E1 & E3 in the outer shell of the complex.

The E3 binding protein (that binds E3 to E2) also has attached lipoamide that can exchange reducing equivalents with lipoamide on E2.

For diagrams showing the approximate arrangement of functional domains, based on structural studies of Pyruvate Dehydrogenase and a related enzyme see: 

  • a website of the laboratory of Wim Hol.
  •  an article by Milne et al. (Fig. 5, requires a subscription to J. Biol. Chem.).

 

 

Organic arsenicals are potent inhibitors of lipoamide-containing enzymes such as Pyruvate Dehydrogenase. These highly toxic compounds react with "vicinal" dithiols such as the functional group of lipoamide as shown at right.

 

 

 

In the overall reaction, the acetic acid generated is transferred to coenzyme A.
The final electron acceptor is NAD+.


 

 

 

The reaction proceeds as follows:

  1. The keto carbon of pyruvate reacts with the carbanion of TPP on E1 to yield an addition compound. The electron-pulling positively charged nitrogen of the thiazole ring promotes loss of CO2. What remains is hydroxyethyl-TPP.
  2. The hydroxyethyl carbanion on TPP of E1 reacts with the disulfide of lipoamide on E2. What was the keto carbon of pyruvate is oxidized to a carboxylic acid, as the disulfide of lipoamide is reduced to a dithiol. The acetate formed by oxidation of the hydroxyethyl moiety is linked to one of the thiols of the reduced lipoamide as a thioester (~).
  3. The acetate is transferred from the thiol of lipoamide to the thiol of coenzyme A, yielding acetyl CoA.
  4. The reduced lipoamide swings over to the E3 active site. Dihydrolipoamide is reoxidized to the disulfide, as 2 e- + 2 H+ are transferred to a disulfide on E3 (disulfide interchange). 
  5. The dithiol on E3 is reoxidized as 2 e- + 2 H+ are transferred to FAD. The resulting FADH2 is reoxidized by electron transfer to NAD+, to yield NADH + H+.

Acetyl CoA, a product of the Pyruvate Dehydrogenase reaction, is a central compound in metabolism. The "high energy" thioester linkage makes it an excellent donor of the acetate moiety.

 

 

For example, acetyl CoA functions as:

Regulation of Pyruvate Dehydrogenase complex  :

 

Product inhibition by NADH and acetyl CoA: NADH competes with NAD+ for binding to E3. Acetyl CoA competes with Coenzyme A for binding to E2.

Regulation by phosphorylation/dephosphorylation of E1: Specific regulatory Kinases and Phosphatases are associated with the Pyruvate Dehydrogenase complex within the mitochondrial matrix.

Pyruvate Dehydrogenase Kinases are activated by NADH and acetyl-CoA, providing another way the two major products of the Pyruvate Dehydrogenase reaction inhibit the complex. Pyruvate Dehydrogenase Kinase activation involves interaction with E2 subunits to sense changes in oxidation state and acetylation of lipoamide caused by NADH and acetyl-CoA.

During starvation, Pyruvate Dehydrogenase Kinase increases in amount in most tissues, including skeletal muscle, via increased gene transcription. Under the same conditions, the amount of Pyruvate Dehydrogenase Phosphatase decreases. The resulting inhibition of Pyruvate Dehydrogenase prevents muscle and other tissues from catabolizing glucose and gluconeogenesis precursors. Metabolism shifts toward fat utilization, while muscle protein breakdown to supply gluconeogenesis precursors is minimized, and available glucose is spared for use by the brain.

A Ca++-sensitive isoform of the phosphatase that removes phosphate residues from E1 is expressed in muscle cells. The increased cytosolic Ca++ that occurs during activation of muscle contraction can lead to Ca++ uptake by mitochondria. The higher Ca++ stimulates the phosphatase, and dephosphorylation activates Pyruvate Dehydrogenase. Thus mitochondrial metabolism may be stimulated during exercise.

Lecture notes relating to Krebs Cycle are not provided in the usual format, because lectures will be presented by students. Some questions on Krebs Cycle are included in the self-study quiz for this class.

Select the interactive tutorial at right for information about the Krebs Citric Acid Cycle. Within the tutorial, drag the cursor over each enzyme name for information about that reaction.

Note that FADH2, listed as a product of succinate oxidation, is reoxidized to FAD as redox carriers within the Succinate Dehydrogenase complex pass electrons to coenzyme Q of the respiratory chain. Thus it would be more appropriate to list coenzyme QH2 as a product of the Succinate Dehydrogenase reaction. The initial acceptor, FAD, is included in the diagram for consistency with most textbooks.

 

 

KREBS CYCLE OR TRICARBOXYLIC ACID CYCLE OR TCA OR CITRIC ACID CYCLE:

 

            This referred as cycle because oxaloacetate regenerated at the end of each cycle and it is also one of the initial substrate enter into the cycle.  This cycle occurs within the mitochondrial matrix.  This is a series of biochemical reactions aerobic organisms use to release chemical energy stored in acetyl CoA.  Acetyl CoA is a product of catabolic reactions in carbohydrate, lipid and amino acid metabolism.  In this cycle, the carbon atoms derived from the acetyl group of acetyl coA are oxidized to CO2.  The high energy electrons removed from citric acid cycle intermediates are transferred to NAD+ and FAD to form the reduced coenzymes NADH and FADH2.  In one of the step, the high energy molecule guanosine triphosphate (GTP) is produced during a substrate level phosphorylation.  The overall reaction for TCA cycle is as follows:

 

 

            Krebs cycle occurs in eight steps.  The detailed reactions of cycle are shown in the figure. 

 

Step 1:

           

            In the first step, acetyl CoA is condensed with oxaloacetate to form citrate.  The reaction is catalyzed by the enzyme citrate synthase. This is reaction is aldol condensation reaction and Citrate formation is highly exergonic because the standard free energy change is equal to - 8 Kcal/mole.    

   

Step 2:

 

            In this step, citrate reversibly converted to isocitrate by aconitase.  During this isomerization reaction, an intermediate called cis-aconitate is formed by dehydration.  The carbon-carbon double bond of cis-aconitate is then rehydrated to form the isomer isocitrate.  This reaction is carried out by aconitase enzyme.

 

Step 3:

 

            In this step, oxidative decarboxylation of isocitrate, catalyzed by isocitrate dehydrogenase, occurs in two steps.  First, isocitrate is oxidized to form oxalosuccinate, a transient intermediate.  Immediate decarboxylation of oxalosuccinate results in the formation of a- ketoglutarate. 

 

            There are two forms of isocitrate dehydrogenase in mammals.  The NAD+ requiring isozyme is found only within mitochondria.  The other isozyme, which requires NADP+, is found in both the mitochondrial matrix and the cytoplasm.  In some circumstances the latter enzyme is used to within both compartments too generate NADPH, which is required in biosynthetic processes.

 

Step 4:

 

            In this step,  a- ketoglutarate is converted into succinyl CoA by the enzyme a - ketoglutarate dehydrogenase complex.  This exergonic, oxidative decarboxylation reaction is analogous to the pyruvate dehydrogenase reaction.  In both reactions, energy rich thioester molecules are products, that is, acetyl CoA and Succinyl CoA.  Other similarities between the two multienzyme complexes are that the same cofactors (TPP, COA-SH, Lipoic acid, NAD+ and FAD) are required and the same or similar allosteric effectors are inhibitors.  In the case of    a- ketoglutarate dehydrogenase, inhibition is produced by succinyl CoA, NADH, ATP and GTP.   An important difference between the two complexes is that the control mechanism of   a- ketoglutarate dehydrogenase does not involve covalent modification. 

 

Step 5:

 

            In this step, Succinyl CoA is converted into Succinate with the formation of GTP through substrate level phosphorylation process.  This reaction is catalyzed by succinate thiokinase.  In mammals GDP is phosphorylated whereas in many other organisms, ADP is phosphorylated instead.

 

Step 6:

 

            In this step, succinate dehydrogenase catalyzes the oxidation of succinate to form fumarate.  Unlike the other citric acid cycle enzymes, succinate dehydrogenase is not found within the mitochondrial matrix.  Instead, it is tightly bound to the inner mitochondrial membrane.  Succinate dehydrogenase is activated by high concentrations of succinate, ATP, and Pi and inhibited by oxaloacetate.  This enzyme also inhibited by malonate which is an structural analog of succinate.

 

Step 7:

 

            In this step, fumarate is converted to L-malate in a reversible stereospecific hydration reaction catalyzed by fumarase which is also otherwise known as fumarate hydratase.

 

Step 8:

 

            In the final step, oxaloacetate is regenerated with the oxidation of L-malate by the enzyme malate dehydrogenase.  Malate dehydrogenase uses NAD+ as the oxidizing agent in a highly endergonic reaction. 

 

 

REGULATION OF TCA :

 

            The cirtric acid cycle enzymes citrate synthase, isocitrate dehydrogenase and   a- ketoglutarate dehydrogenase are closely regulated because they catalyze reactions that represent important metabolic branch points. 

 

Citrate synthase:

 

            This enzyme is regulated by the concentration of substrate oxaloacetate and acetyl CoA.  Because of acetyl CoA and oxaloacetate are low in mitochondria in relation to the amount of the enzyme, any increase in substrate availability stimulates citrate synthesis.  High concentrations of succinyl CoA and citrate inhibit citrate synthase by acting as allosteric inhibitors.  Other allosteric regulators of this reaction are NADH and ATP, whose concentrations reflect the cellís current energy status.  A resting cell has high NADH / NAD + and ATP / ADP ratios.  As a cell becomes metabolically active, NADH and ATP concentrations decrease.  Consequently, key enzymes such as citrate synthase become more active.

 

Isocitrate Dehydrogenase:

 

            Isocitrate dehydrogenase catalyzes the second closely regulated reaction in the cycle.  Its activity is stimulated by relatively high concentrations of ADP and NAD + and inhibited by ATP and NADH.  This enzyme is closely regulated because of its important role in citrate metabolism.

 

 a - ketoglutarate Dehydrogenase:

 

            This enzyme is strictly regulated because of the important role of   a - ketoglutarate in several metabolic processes.  When a cellís energy stores are low,  of   a - ketoglutarate dehydrogenase is activated and of   a - ketoglutarate is retained within the cycle at the expense of biosynthetic processes.  As the cellís supply of NADH rises, the enzyme is inhibited and of   a - ketoglutarate molecules become available for biosynthetic reactions.

 

            Fluroacetate can inhibit aconitase and then inhibit TCA cycle. 

 

TCA IS AMPHIOBOLIC:

 

            The citric acid cycle is obviously catabolic, since acetyl groups are oxidized to form CO2 and energy is conserved in reduced coenzyme molecules.  The citric acid cyle is also anabolic, since several citric acid cycle intermediates are precursors in biosynthetic pathway.  For example, oxaloacetate is used in both gluconeogenesis and aminoacid synthesis.     a - ketoglutarate also plays an important role in aminoacid synthesis.  The synthesis of porphyrins such as heme uses succinyl CoA.  Finally, the synthesis of fatty acids and cholesterol in the cytoplasm requires acetyl CoA.  Because acetyl CoA cannot penetrate in the inner mitochondrial membrane, it is converted to citrate.  After its transport into the cytoplasm, citrate is cleaved to form acetyl CoA and oxaloacetate by citrate lyase.

 

            Anaplerotic reaction, a reaction that replenishes a substrate needed for a biochemical pathway, available to replenish the intermediates of citric acid cycle.  One of the most important anaplerotic reactions is catalyzed by pyruvate carboxylase.  A high concentration of acetyl CoA, an indicator of an insufficient oxaloacetate concentration, activates pyruvate carboxylase.  As a result, oxaloacetate concentration increases.  Any excess oxaloacetate that is not used within the citric acid cycle is used in gluconeogenesis.  Other anaplerotic reactions include the synthesis of succinyl CoA from certain fatty acids and certain amino acids.

 

Glyoxylate Cycle :

            In general, the Krebs cycle functions similarly in bacteria and eukaryotic systems, but major differences are found among bacteria. One difference is that in obligate aerobes, L-malate may be oxidized directly by molecular 02 via an electron transport chain. In other bacteria, only some Krebs cycle intermediate reactions occur because a-ketoglutarate dehydrogenase is missing.

            A modification of the Krebs cycle, commonly called the glyoxylate cycle, or shunt , which exists in some bacteria. This shunt functions similarly to the Krebs cycle but lacks many of the Krebs cycle enzyme reactions. The glyoxylate cycle is primarily an oxidative pathway in which acetyl~SCoA is generated from the oxidation, of acetate, which usually is derived from the oxidation of fatty acids. The oxidation of fatty acids to acetyl~SCoA is carried out by the b-oxidation pathway. Pyruvate oxidation is not directly involved in the glyoxylate shunt, yet this shunt yields sufficient succinate and malate, which are required for energy production . The glyoxylate cycle also generates other precursor compounds needed for biosynthesis . The glyoxylate cycle was discovered as an unusual metabolic pathway during an attempt to learn how lipid (or acetate) oxidation in bacteria and plant seeds could lead to the direct biosynthesis of carbohydrates. The glyoxylate cycle converts oxaloacetate either to pyruvate and CO2 (catalyzed by pyruvate carboxylase) or to phosphoenolpyruvate and CO2 (catalyzed by the inosine triphosphate [ITP]-dependent phosphoenolpyruvate carboxylase kinase). Either triose compound can then be converted to glucose by reversal of the glycolytic pathway. The glyoxylate cycle is found in many bacteria, including Azotobacter vinelandii and particularly in organisms that grow well in media in which acetate and other Krebs cycle dicarboxylic acid intermediates are the sole carbon growth source. One primary function of the glyoxylate cycle is to replenish the tricarboxylic and dicarboxylic acid intermediates that are normally provided by the Krebs cycle. A pathway whose primary purpose is to replenish such intermediate compounds is called anaplerotic.