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PYRUVATE DEHYDROGENASE AND THE KREBS CYCLE

Pyruvate is converted to acetyl-CoA plus CO2 by pyruvate dehydrogenase and the acetyl-CoA is converted to two CO2 by the Krebs cycle. Both produce reduced NADH and also use the cofactor FAD. We will consider these two cofactors first.

COFACTORS FOR REDOX REACTIONS

1) NAD and NADP = Nicotinamide adenine dinucleotide (phosphate). Previously called DPN and TPN or coenzymes I and II. Nicotinic Acid, of which nicotinamide is the amide, is also called niacin (when added to cornflakes, etc.). These two coenzymes are known as the pyridine nucleotides and function as coenzymes for many dehydrogenases. Most dehydrogenases take NAD or NADP, but not both, and NAD is rather more common than NADP. Usually NAD removes 2H in catabolism, whereas NADPH2 donates 2H in biosynthesis. For example:

Although NAD carries two reducing equivalents, only one hydrogen atom attaches to the nicotinamide ring. The second hydrogen atom becomes a hydrogen ion in solution. This is written:

2) FMN and FAD. Like NAD/NADP, these coenzymes are usually bound non-covalently by their enzymes. (Occasional exceptions do occur). FMN is flavin mononucleotide (= riboflavin phosphate) and FAD is flavin adenine dinucleotide. FAD is FMN linked via a phosphate to AMP. The oxidized forms are colored yellow, red or green, the reduced forms are bleached. Both hydrogens derived from a redox reaction become attached to the flavin ring.

FAD + 2(H) = FADH2

PYRUVATE DEHYDROGENASE

If oxygen is present, reoxidation of NADH is not a problem. Pyruvate is decarboxylated to acetyl-CoA by the pyruvate dehydrogenase complex and the acetyl-CoA is oxidized by the Krebs cycle. Overall reaction:

The pyruvate dehydrogenase complex consists of three enzymes:

E1, pyruvate decarboxylase, splits pyruvate into CO2 and a 2-carbon fragment which is attached to its cofactor - thiamine pyrophosphate (TPP). The 2-carbon fragment is attached to the five membered ring and replaces the hydrogen atom which is circled in the diagram.

In certain organisms, e.g. yeast, pyruvate decarboxylase acts independently during fermentation and releases the 2-carbon fragment as acetaldehyde so converting pyruvate to acetaldehyde plus CO2. In aerobic growth, it acts as part of the PDH complex. After releasing CO2 it hands on the 2-carbon fragment (the hydroxyethyl group) to E2, the next enzyme in the pyruvate dehydrogenase complex (see diagram).

E2, lipoate transacetylase, receives the two carbon fragment. The 2-carbon fragment is attached to one of the sulfurs on the lipoic acid cofactor, and the S-S bond is opened up. Lipoic acid is covalently bound to E2 via the side-chain amino group of a lysine residue: Lipoic acid-CO-NH-lysine-E2

The two carbon fragment is then transferred to the sulfhydryl group of coenzyme A. Coenzyme A is a universal carrier of acyl groups (see diagram). CoA consists of adenosine monophosphate (adenine, ribose, phosphate) plus an extra phosphate (attached to the 3' hydroxyl of the ribose), linked to phosphopantetheine (phosphate, pantothenic acid, cysteamine). Cysteamine is derived by decarboxylation from cysteine and provides the active sulfhydryl group.

E3, lipoamide dehydrogenase, carries an FAD cofactor which reoxidizes the lipoic acid of E2. The pyruvate dehydrogenase complex consists of a core of E2 to which the other enzymes are attached. The long lipoic acid-lysine side chain of E2 acts as a swinging arm to convey the C2 fragment from E1 to CoASH and also carries the lipoate residue past the active site of E3 for reoxidation by the FAD cofactor.

Finally, the E3-FADH2 is reoxidized to E3-FAD by transfer of its reducing equivalents to NAD:

E3 - FADH2 + NAD+ = NADH + H+ + E3-FAD

In eukaryotes, pyruvate dehydrogenase is regulated by covalent modification (addition or removal of a phosphate group) - as stated in most textbooks. In bacteria this does not happen. Instead there are two other types of regulation:

a) Genetic - the PDH operon (including the aceE, aceF and lpd genes) is induced by pyruvate. The PdhR repressor protein switches the operon off when pyruvate is absent. If pyruvate is present, it binds to and inactivates the PdhR protein.

b) Enzyme activity - high levels of NADH inhibit this enzyme (NADH builds up during anaerobic conditions, especially fermentation).

THE KREBS CYCLE

The acetyl-CoA now enters the Krebs cycle (also known as the citric acid cycle or tricarboxylic acid cycle). The NADH made by PDH, together with the NADH made by the Krebs cycle itself, is reoxidized by the respiratory chain (see next section).

Krebs won the Nobel Prize by realizing that a linear pathway was no good - he proposed the cycle. (Krebs also invented the Urea Cycle in higher organisms--everyone else kept trying to fit the reactions into linear schemes again.) Evidence for the Krebs cycle:

ENZYME	Cofactors	Inhibitors	DG (kcal/mol)
1. Citrate synthase			-9.1
2. Aconitase		fluorocitrate	+1.6
3. Isocitrate dehydrogenase	NADP		-1.7
4. a-Ketoglutarate dehydrogenase	NAD, FAD, lipoate	arsenite	-8.8
5. Succinic thiokinase	ATP	hydroxylamine	-2.1
6. Succinate dehydrogenase	FAD, FeS group	malonate	0
7. Fumarase		meso-tartrate,	-0.9
8. Malate dehydrogenase	NAD	b-fluoroxalacetate fluoromalate	+6.7

Arsenite reacts with lipoic acid and inactivates it. Hydroxylamine reacts with succinyl-CoA to give the hydroxamate of succinic acid. The other inhibitors are competitive inhibitors i.e. analogs of the substrates.

The individual reactions of the Krebs Cycle:

1. Citrate synthase

acetyl-CoA + oxaloacetate = citrate + CoASH

ATP acts as an allosteric inhibitor which reduces the affinity of the enzyme for acetyl-CoA. Hence surplus energy slows down the Krebs Cycle. Citrate synthase will convert fluoroacetyl-CoA into fluorocitrate which is extremely toxic because it inhibits aconitase. Fluoroacetyl-CoA is made from fluoroacetate by acetate kinase and phosphotransacetylase. The conversion of a substance such as fluoroacetate, which is itself "harmless", to a toxic compound is known as a lethal synthesis. Fluoroacetate is one of the most toxic chemicals known. It is found in certain plants and used as a poison for blow-pipe darts by certain South American Indians.

2. Aconitase

citrate = cis-aconitate = isocitrate

H2O is removed to give cis-aconitate and then added back to give iso-citrate. Fe2+ is required and the pure enzyme is very oxygen sensitive.

3. Isocitrate dehydrogenase

I) iso-citrate + NADP+ = oxalosuccinate + NADPH

II) oxalosuccinate = alpha-ketoglutarate + CO2

The oxalosuccinate is an enzyme bound intermediate. In bacteria NADP is reduced whereas in eukaryotes NAD is used.

4. alpha-Ketoglutarate dehydrogenase complex.

[Note ketoglutarate and oxoglutarate are different names for the same substance.] Consists of 3 enzymes, analogous to the 3 components of the pyruvate dehydrogenase complex. The oxoglutarate multienzyme complex is smaller than the pyruvate complex because it has fewer of each subunit.

In E. coli and mitochondria ATP is made. In the cytoplasm of animals GTP is produced as is usually stated in textbooks. This step is substrate level phosphorylation. A histidine bound phosphate is an intermediate.

I) Succinyl-CoA + Pi = Succinyl-P(enzyme bound) + CoASH

II) Enz + Succinyl-P = Succinate + Enz-histidine-P

III) Enz-histidine-P + ADP = Enz + ATP

6. Succinate dehydrogenase

Succinate + FAD = Fumarate + FADH2

SDH is tightly bound to the membrane in close association with the electron transport chain. SDH is activated by succinate and ATP and inhibited by oxaloacetate. It contains FAD and Fe-S (non-heme-iron) groups. There are 4 protein subunits:

A: Enzyme subunit - carries the FAD and 4 Fe-S groups.

B: Electron transfer subunit - carries another 4 Fe-S groups and transfers electrons to the electron transport chain

C and D: Small hydrophobic membrane proteins which anchor A and B to the membrane.

Although energetically unfavorable it goes forward because NADH is oxidized rapidly via the respiratory chain and oxaloacetate goes on to react with another acetyl-CoA molecule.

Products and Energetics of Krebs cycle

For each acetyl-CoA that enters the Krebs cycle we get:

2 CO2

1 ATP

1 FADH2 = 2 ATP

3 NAD(P)H = 3 ATP each = 9 ATP

Sum = 12 ATP/acetyl-CoA = 24 ATP/glucose

Glycolysis gives 2 ATP/glucose plus 2 NADH (= 6 ATP)/glucose

Pyruvate dehydrogenase gives 2 NADH (= 6 ATP)/glucose

Grand total = 38 ATP/glucose

ANAPLEROTIC SEQUENCES

Anaplerotic pathways - replenish intermediates.

If anabolic pathways (e.g. for synthesis of amino acids) consume an intermediate from a cyclic pathway (e.g. the Krebs cycle) then the cycle will grind to a halt if nothing is done to replace it. Anaplerotic pathways replenish the intermediates of the Krebs cycle (and other cyclic pathways). We will restrict ourselves to anaplerotic pathways that keep the level of Krebs cycle intermediates constant (or elevate them if needed).

The Krebs Cycle is subject to a major drain of intermediates by the conversion of oxaloacetate to aspartic acid (needed for making Asn, Met, Lys, Thr and Ile) and of ketoglutarate to glutamic acid (for Gln, Arg, Pro). Remember, the acetyl-CoA which enters the Krebs cycle is all turned into CO2. Contrast this with the synthesis of alanine from pyruvate or of serine from 3-phosphoglyceric acid which pose no problem because glycolysis is a linear pathway and new intermediates are constantly made from glucose.

The problem is solved by converting a 3-carbon glycolytic intermediate to a 4-carbon Krebs cycle intermediate by the fixation of CO2. PEP Carboxylase (PPC) is the major route in most bacteria including E. coli:

Phosphoenolpyruvate + CO2 = oxaloacetate + Pi

Mutants of E. coli which lack PEP Carboxylase (ppc) do not grow on glucose, glycerol, pyruvate or other carbon sources which lead to these as intermediates, unless a 4-carbon compound is supplied. In eukaryotes (yeast and animals) and in some bacteria e.g. Pseudomonas, pyruvate carboxylase is used instead:

Pyruvate + CO2 + ATP = oxaloacetate + ADP + Pi

If E. coli is growing on succinate or other Krebs cycle intermediates then it faces the reverse problem. It must make 3-carbon metabolites from 4-carbon compounds. Two reactions are involved. PEP carboxykinase produces PEP which is needed to make glycolytic intermediates and sugars:

If E. coli grows on acetate or other carbon sources (e.g. fatty acids) which feed directly into acetyl-CoA it cannot convert PEP to oxaloacetate since it has no PEP to convert. Growth on two carbon substrates requires the glyoxylate bypass. Two extra enzyme are required:

Isocitrate lyase (ICL): isocitrate = succinate + glyoxylate

Malate synthase (MAS): glyoxylate + acetyl-CoA = malate + CoA

If we combine these reactions with that part of the Krebs cycle which runs from malate to isocitrate, including the incorporation of acetyl CoA, the overall result is the conversion of two acetyl-CoA's to a 4-carbon acid (see diagram - heavy lines are extra reactions). The decarboxylation steps between isocitrate and succinate are omitted. The combination of ICL, MAS and this part of the Krebs cycle is known as the Glyoxylate cycle.

The enzymes ICL, MAS and a third enzyme, ICDH kinase, are all induced by growth on acetate or fatty acids and repressed in the presence of sugars, Krebs cycle intermediates, and almost every other carbon source. The aceB gene encodes malate synthase, aceA encodes isocitrate lyase and aceK encodes ICDH kinase. The aceBAK operon is controlled by the IclR repressor protein but the nature of the metabolite which acts as inducer is still unknown.

During growth on acetate, ICDH kinase attaches a phosphate group to about 75% of the cell's isocitrate dehydrogenase (ICDH). This inactivates the isocitrate dehydrogenase because the phosphate blocks the active site and its negative charge repels the isocitrate which is also negatively charged. ICDH needs to be partly inactivated since otherwise it would use all the isocitrate as it has a much greater affinity for isocitrate than does ICL. The ICDH kinase can also remove the phosphate group, depending on the circumstances:

a) When the level of isocitrate or of 3-P-glycerate falls this signals a lack of biosynthetic intermediates and phosphate groups are added to ICDH which is therefore inactivated.

b) If the level of pyruvate rises (because a good carbon source is now available) phosphate groups are removed and ICDH is reactivated.

c) If the level of AMP rises, this signals energy starvation and means we need to oxidize more acetyl-CoA by running the normal Krebs cycle reactions. So phosphate groups are removed to re-activate ICDH.

Control of enzyme activity by covalent modification is rare in prokaryotes but relatively common in higher organisms.

Biosynthesis requires large amounts of NADPH. Sources include:

1) energy linked transhydrogenase (see below)

2) isocitrate dehydrogenase is NADP-linked in bacteria

3) the NADP-linked malic enzyme

4) the hexose monophosphate shunt

The Entner-Doudoroff (ED) pathway is intended for the degradation of sugar acids (e.g. glucuronic, galacturonic and gluconic). It is sometimes called the hexose monophosphate pathway to distinguish it from the normal glycolytic pathway (= hexose diphosphate = Embden-Meyerhof or EM pathway). The Pentose-Phosphate cycle is intended for the interconversion of pentoses and hexoses. The hexose monophosphate shunt (HMP-shunt) links together the "starting" points of the Embden-Meyerhof, Entner-Doudoroff and Pentose-P pathways.

Versatile organisms like E. coli possess all three pathways. In E. coli 80% of the glucose goes via the EM pathway. The other 20% is sent via the HMP shunt and through the pentose pathway. The purpose of this manoeuvre is to generate reduced NADPH. Both the glucose-6-P dehydrogenase and the HMP shunt and the gluconate-6-P dehydrogenase are linked to NADP (not NAD).

Next the 6-phosphogluconolactone is interconverted with gluconate-6P by phosphogluconolactonase.

In E. coli the ED pathway is used for growth on hexuronic acids (e.g. glucuronic acid). In Pseudomonas and its relatives like Zymomonas, there is no Embden-Meyerhof glycolytic pathway and all of the glucose goes via the HMP shunt into the ED pathway and is degraded this way.

Key to Shunt/Pentose/E-D Diagram	Gene	Map Position
The Hexose MonoPhosphate shunt:
1. glucose-6P dehydrogenase (zwischenferment)	zwf	41
2. phosphogluconolactonase	pgl	17
3. gluconate-6P dehydrogenase	gnd	44
5. phosphogluconate dehydratase (Entner-Doudoroff dehydratase)	edd	41
Gluconate metabolism:
4. gluconokinases (two)	gntM,S	75, 95
Entner-Doudoroff pathway:
5. (see above) sometimes included in ED pathway	edd	41
6. ketodeoxygluconate kinase	kdgK	78
7. ketodeoxygluconate-P aldolase (Entner-Doudoroff aldolase)	eda	41
Pentose cycle:
3. (see above) sometimes considered part of oxidative pentose cycle	gnd	44
8. phosphoketopentose epimerase	?	?
8. ribulose-P epimerase (arabinose specific)	araD	1
9. ribose-P isomerase	rpiA	62
10. transketolase (2 reactions, same enzyme).	tkt	62
11. transaldolase	tal	?

Note: GA3P = glyceraldehyde-3-P, KDG = ketodeoxygluconate

The pentose phosphate cycle

The pentose-P pathway can operate in two alternative modes:

The oxidative version of the pentose cycle includes the gluconate-6P dehydrogenase reaction, which is usually regarded as part of the hexose monophosphate shunt.

This pathway is normally used to make pentoses - like the ribose and deoxyribose present in RNA and DNA. E. coli can also use the non-oxidative mode of the pentose cycle to grow on five carbon sugars as carbon sources. These are converted to hexose and then enter the EM pathway. In practice the "hexose" is produced as a mixture of fructose-6P and glyceraldehyde-3P and thus enters the EM pathway half way down.