February 15 1 Dentistry Medicine

February 15 2 Medicine

Pyruvate Dehydrogenase

Pyruvate

dehydrogenase Dihydrolipoyl

dehdrgenase

Dihydrolipoyl

transacetylase

TPP FAD Lipoic

February 15 3

irreversible;

in mitochodria.

Medicine

Pyruvate dehydrogenase complex:

E1 pyruvate dehydrogenase

Es E2 dihydrolipoyl transacetylase

E3 dihydrolipoyl dehydrogenase

Thiamine pyrophosphate, TPP (VB1)

Cofactors Lipoic Acid

(Vitamins) CoA (pantothenic acid)

FAD (VB2)

NAD+ (VB3)

Tender

Loving

Care

For

Nancy

Medicine

mnemonics

Pyruvate Dehydrogenase

Subunits

Enzyme Abbreviated Prosthetic Group

Pyruvate

Dehydrogenase E1

Thiamine

pyrophosphate (TPP)

Dihydrolipoyl

Transacetylase E2

Lipoamide

Dihydrolipoyl

Dehydrogenase E3

FAD

Medicine

February 15 7 Medicine

The lipoyllysyl moiety is the prosthetic group of dihydrolipoyl transacetylase (E2 of the

PDH complex). The lipoyl group occurs in oxidized (disulfide) and reduced (dithiol)

forms and acts as a carrier of both hydrogen and an acetyl (or other acyl) group.

Lipoic acid (lipoate) in amide linkage with a Lys residue

February 15 8 Medicine

Localization of pyruvate decarboxylation

The transport of pyruvate into the mitochondria is via a transport protein and is active, consuming energy. Passive diffusion of pyruvate into the mitochondria is impossible because it carries a negative charge.

On entry to the mitochondria the pyruvate decarboxylation occurs, producing acetyl CoA. This irreversible reaction traps the acetyl CoA within the mitochondria (the acetyl-CoA can only be transported out of the mitochondrial matrix under conditions of high oxaloacetate via the citrate shuttle, a TCA intermediate). The carbon dioxide produced by this reaction is nonpolar and small, and can diffuse out of the mitochondria and out of the cell.

Medicine

Citrate Shuttle Acetyl-CoA can only be transported out of the mitochondrial matrix under conditions of high

oxaloacetate via the citrate shuttle

Medicine

Oxidative decarboxylation of pyruvate to acetyl-CoA by the PDH complex

The first step is the slowest and therefore limits the rate of the overall reaction.

It is also the point at which the PDH complex exercises its substrate specificity February 15 13 Medicine

Medicine

Oxidative decarboxylation of

pyruvate.

Regulation of Pyruvate

Dehydrogenase Complex:

NADH competes with NAD+ for binding to E3.

Acetyl CoA competes with CoA for binding to E2.

Pyruvate (-)

Pyruvate dehydrogenase is inhibited when one or more of the three following ratios are increased: ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA.

In eukaryotes PDC is tightly regulated by its own specific pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP), deactivating and activating it respectively.

PDK phosphorylates three specific serine residues on E1 with different affinities. Phosphorylation of any one of them renders E1 (and in consequence the entire complex) inactive.

Dephosphorylation of E1 by PDP reinstates complex activity. Products of the reaction act as allosteric inhibitors of the PDC, because

they activate PDK. Substrates in turn inhibit PDK, and thus, reactivating PDC.

During starvation, PDK increases in amount in most tissues, including skeletal muscle, via increased gene transcription. Under the same conditions, the amount of PDP decreases. The resulting inhibition of PDC 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.

Calcium ion has a role in regulation of PDC in muscle tissue, because it activates PDP, stimulating glycolysis on its release into the cytosol - during muscle contraction.

Medicine

(1) Pyruvate dehydrogenase

complex

Pyruvate dehydrogenase(active form)

allosteric inhibitors:

ATP, acetyl CoA,NADH, FA

allosteric activators:

AMP, CoA,

NAD+,Ca2+

pyruvate dehydrogenase (inactive form)

P

pyruvate dehydrogenase kinase

pyruvate dehydrogenase phosphatase

ATP

ADPH2O

Pi

Ca2+,insulin acetyl CoA,NADH

ADP,

NAD+Medicine

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 & gluconeogenesis precursors.

Metabolism shifts toward fat utilization.

Muscle protein breakdown to supply gluconeogenesis precursors is increased.

Available glucose is spared for use by the brain.

Medicine

Advantages of multienzyme complex:

The PDH complex is a classic multienzyme complex in which a series of chemical intermediates remain bound to the enzyme molecules as a substrate is transformed into the final product. Five cofactors, four derived from vitamins, participate in the reaction mechanism. The regulation of this enzyme complex also illustrates how a combination of covalent modification and allosteric regulation results in precisely regulated flux through a metabolic step

Higher rate of reaction: Because product of one enzyme acts as a substrate of other, and is available for the active site of next enzyme without much diffusion.

Minimum side reaction.

Coordinated control.

Medicine

Treatment Use of a ketogenic diet has been described. The ketogenic diet is a high-fat, adequate-protein, low-carbohydrate

diet. The diet mimics aspects of starvation by forcing the body to burn fats rather than carbohydrates.

Current research is being conducted on the viability of Dichloroacetic acid to treat the lactic acidosis commonly accompanied by this disorder. Salts of DCA have been studied as potential drugs because they stimulates the activity of the enzyme pyruvate dehydrogenase by inhibiting the enzyme pyruvate dehydrogenase kinase. Thus, it decreases lactate production by shifting the metabolism of pyruvate from glycolysis towards oxidation in the mitochondria.

Additionally, there is research being conducted on the viability of gene therapy for sufferers of this condition as well as many other mitochondrial defects.

Medicine

Medicine

Thiamine is involved in a vast array of functions:

Carbohydrate metabolism Production of the neurotransmitters glutamic

acid and GABA, through the citric acid cycle

Lipid metabolism, necessary for myelin production Amino acid metabolism Neuromodulation - physiological process by which a

given neuron uses one or more neurotransmitters to

regulate diverse populations of neurons.

Thiamin (Vitamin B1) deficiency causes Beriberi

Thiamine pyrophosphate (TPP) is an important cofactor of pyruvate dehydrogenase complex, or PDC a critical enzyme in glucose metabolism. Thiamine is neither synthesized nor stored in good amounts by most vertebrates. It is required in the diets of most vertebrates.

The body cannot produce thiamine and can only store up to 30 mg of it in tissues. Thiamine is mostly concentrated in the skeletal muscles. Other organs in which it is found are the brain, heart, liver, and kidneys. The half-life of thiamine is 9-18 days. It is excreted by the kidney

Thiamine deficiency ultimately causes a fatal disease called Beriberi characterized by neurological disturbances, paralysis, atrophy of limbs and cardiac failure. Note that brain exclusively uses aerobic glucose catabolism for energy and PDC is very critical for aerobic catabolism. Therefore thiamine deficiency causes severe neurological symptoms.

The main types of beriberi are:

Dry beriberi and Wernicke-Korsakoff syndrome affect the peripheral and central nervous system respectively.

Wet (edematous) beriberi affects the cardiovascular system, as well as other bodily systems. Infantile beriberi affects mostly children in developing countries

Prevalence

Beriberi is rare in developed countries because most foods are now vitamin-enriched. Excluding the presence of arsenic in the environment (e.g. well water) one can get enough thiamine by eating a normal, healthy diet. Today, beriberi occurs mostly in patients who abuse alcohol. Drinking heavily can lead to poor nutrition, and excess alcohol makes it harder for the body to absorb and store thiamine.

Medicine

Insufficient thiamine significantly impairs glucose oxidation, causing highly aerobic tissues, such as brain and cardiac muscle, to fall first. In addition, branched-chain amino acids are sources of energy in brain and muscle

Wernicke-Korsakoff syndrome is a brain disorder due to thiamine deficiency.

Wernickes Encephalopathy (peripheral neuropathy ) and Korsakoffs Psychosis are the acute and chronic phases, respectively, of the same disease.

Korsakoff syndrome, or Korsakoff psychosis, tends to develop as Wernicke's symptoms go away. Wernicke's encephalopathy causes brain damage in lower parts of the brain called the thalamus and hypothalamus. Korsakoff psychosis results from damage to areas of the brain involved with memory.

The disease is typically associated with chronic alcoholism, but may be associated with malnutrition or other conditions which cause nutritional deficiencies. Alcohol interferes with thiamine absorption from the intestine,

Congestive heart failure may be a complication (wet beri-beri) owing to inadequate ATP and accumulation of ketoacids in the cardiac muscles. (Peripheral vasodilation leading to a high cardiac output state. This leads to salt and water retention and Edema.)

Two other enzyme complexes similar to pyruvate dehydrogenase that use thiamine are:

-Ketoglutarate dehydrogenase (citric acid cycle) Branched-chain ketoacid dehydrogenase (metabolism of branched-chain amino acids)

.

Wernicke-Korsakoff Syndrome (WKS)

Medicine

Arsenic Poisoning: Arsenic compounds such as arsenite (AsO3---) organic arsenicals are poisonous because they covalently bind to sulfhydryl compounds

(SH- groups of proteins and cofactors). Dihydrolipoamide is a critical cofactor of

PDC, and it has two-SH groups, which are important for the PDC reaction. These

SH groups are covalently inactivated by arsenic compounds as shown below;

OH HS S

-O As + -O As + 2H2O

OH HS S

R R

Arsenic compounds in low doses are very toxic to microorganisms, therefore

these compounds were used for the treatment of syphilis and other diseases in

earlier days. Arsenicals were first antibiotics, but with a terrible side effects as

they are eventually very toxic to humans.

Unfortunately and ignorantly, a common nineteenth century tonic, the Fowlers solution contained 10 mg/ml arsenite. This tonic must have been responsible for

many deaths, including the death of the famous evolution scientist Charlse

Darwin.

Medicine

Medical Students

Glycolysis in disease

Genetic diseases

Glycolytic mutations are generally rare due to

importance of the metabolic pathway, this

means that the majority of occurring mutations

result in an inability for the cell to respire, and

therefore cause the death of the cell at an early

stage. However, some mutations are seen with

one notable example being Pyruvate kinase

deficiency, leading to chronic hemolytic anemia.

Medical Students

Genetic defects of this enzyme cause the disease known as pyruvate kinase deficiency.

In this condition, a lack of pyruvate kinase slows down the process of glycolysis.

This effect is especially devastating in cells that lack mitochondria, because these cells must use anaerobic glycolysis as their sole source of energy because the TCA cycle is

not available.

One example is red blood cells, which in a state of pyruvate kinase deficiency rapidly become deficient in ATP and can undergo hemolysis. Therefore, pyruvate kinase

deficiency can cause hemolytic anemia and an increase in plasma bilirubin.

A discrepancy between red blood cell energy requirements and ATP generating capacity produces irreversible membrane injury resulting in cellular distortion, rigidity,

and dehydration. This leads to premature erythrocyte destruction by the spleen and

liver.

Pyruvate Kinase Deficiency

Medical Students

The buildup of reaction intermediates can also increase the level of 2,3-bisphosphoglycerate in the cells and affect tissue oxygenation.

This will cause a "right shift" in the hemoglobin oxygen saturation

curve, implying a decreased oxygen affinity for the hemoglobin and

earlier oxygen unloading than under normal conditions.

Low prevalence, No Heinz bodies (inclusions or aggregates within red blood cells composed of denatured hemoglobin )

Second cause of hemolytic anemia after G6Pdase deficiency.

Treatment can include a blood transfusion or removal of the spleen. Treatment is usually effective in reducing the severity of the

symptoms.

Pyruvate Kinase Deficiency

Medical Students

2-3-BPG BIND TO Hb, DECREASE ITS AFFINITY TO O2, INCREASE O2

AVALABILITY TO TISSUE.

In RBC

Medical Students

o2O2 PRESSURE (torr)

SATURATION

1

0

10 50

No BPG

With BPG

BPG Lowers the binding affinity of Hb for O2

[BPG] = 0, Hb P50 = 1 torr [BPG] = 4000mM, Hb P50 = 26 torr

Without BPG, Hb couldnt unload O2 in cells

Hb

Medical Students

Glycolysis as an indicator of

disease

The glycolytic rates in malignant, rapidly-growing tumor cells are up to 200 times higher than those of their normal tissues of origin, despite the ample availability ofoxygen. A classical explanation holds that the local depletion of oxygen within the tumor is the cause of increased glycolysis in these cells. However, there is also strong experimental evidence that attributes these high rates to an over-expressed form of the enzyme hexokinase (Bustamante and Pedersen 2005),which is responsible for driving the high glycolytic activity when oxygen is not necessarily depleted. This finding currently has an important medical application: aerobic glycolysis by malignant tumors is utilized clinically to diagnose and monitor treatment responses of cancers using medical imaging techniques (Pauwels et al. 2000, PETNET Solutions 2006).

Medical Students

Ethanol Metabolism and Gluconeogenesis

Ethanol strongly inhibits gluconeogenesis and can bring about hypoglycemia, a potentially dangerous decrease in blood glucose levels. Ethanol metabolism occurs primarily in the liver. The reaction, catalyzed by alcohol dehydrogenase is shown as follows:

Ethanol + NAD+ Acetaldehyde + NADH + H+

The NADH produced in this reaction shifts the equilibrium in the liver cytosol of the lactate dehydrogenase from pyruvate formation to lactate synthesis.

The NADH also favors reduction of oxaloacetate to malate in the reaction catalyzed by malate dehydrogenase, making less oxalacetate available forgluconeogenesis. The resulting hypoglycemia can affect the part of the brain concerned with temperature regulation and the body temperature can fall by as much as 2 C. Thus, feeding alcohol to people suffering from hypothermia is counterproductive. Metabolically speaking, glucose would be far more effective in raising body temperature.

Medical Students

Hereditary fructose-

1,6-bisphosphatase

deficiency results in

severely impaired

hepatic

gluconeogenesis and

leads to episodes of

hypoglycemia, apnea,

hyperventillation,

ketosis and lactic

acidosis.

FRUCTOSE METABOLISM

Sequestering ATP

Medical Students

GALACTOSE METABOLISM

SORBITOL/

Cataracts early in life

Cataracts early in life.

Vomiting, diarrhea following lactose ingestion.

Lethargy.

Liver damage, hyperbilirubinemia.

Mental retardation.

Gal-1-P-uridyltransferase deficiency:

Sequestering ATP

Medical Students

Figure 12.4. Sorbitol metabolism

Medical Students

Conversion of glucose to fructose via sorbitol Synthesis of sorbitol:

~Aldose reductase reduces glucose, producing sorbitol (glucitol).

~This enzyme is found, in lens, retina, Schwann cells, liver, kidney,

placenta, red blood cells, and in cells of the ovaries and seminal

vesicles

~In cells of the liver, ovaries, sperm, and seminal vesicles, there is a

second enzyme, sorbitol dehydrogenase, that can oxidize the sorbitol to produce fructose sperm cells use fructose .

~pathway from sorbitol to fructose in the liver provides a mechanism

by which any available sorbitol is converted into a substrate that can

enter glycolysis or gluconeogenesis.

Medical Students

The effect of hyperglycemia on sorbitol metabolism:

Because insulin is not required for the entry of glucose into the

cells where sorbitol syn. occurs. large amounts of glucose may

enter during hyperglycemia (uncontrolled diabetes).

Elevated intracellular glucose produce a significant increase in the

amount of sorbitol, which cannot pass efficiently through cell

membranes and, therefore, remains trapped inside the cell.

This is exacerbated when sorbitol dehydrogenase is low or absent(in retina, lens, kidney, and nerve cells).

result, sorbitol accumulates in these cells(strong osmotic effects

(cataract formation, peripheral neuropathy, and vascular problems

leading to nephropathy and retinopathy)

Medical Students

2. The effect of hyperglycemia on sorbitol metabolism

- Because insulin is not required for entry of gluc into cells listed in previous paragraph, large amounts of gluc may enter these cells during times of hyperglycemia, e.g., in uncontrolled diabetes.

- Elevated intracellular gluc concs & an adequate supply of NADPH cause aldose reductase to produce a sufficient increase in the amount of sorbitol, which cant pass efficiently through CMs &, therefore, remains trapped inside cell.

- This is exacerbated when sorbitol dehydrogenase is low or absent, e.g., in retina, lens, kidney & nerve cells. As a result, sorbitol accumulates in these cells, causing strong osmotic effects &, therefore, cell swelling as a result of water retention

- Some of the pathologic alterations associated with diabetes can be attributed, in part, to this phenomenon, including cataract formation, peripheral neuropathy, & vascular problems leading to nephropathy, & retinopathy