Lecture 18-Pyruvate Dehydrogenase Complex

Describe how and where pyruvate oxidation occurs in mammalian cells.

Pyruvate Oxidation Occurs in the Mitochondrion

Under aerobic conditions, in eukaryotes, the pyruvate generated from glucose is transported into the mitochondrial matrix for oxidation via the mitochondrial pyruvate carrier (MPC). This is an example of compartmentalization, a form of regulation of metabolic processes that we talked about previously.

Pyruvate Oxidation Generates...

Acetyl CoA

Under aerobic conditions, the pyruvate generated from glucose is converted to acetylCoA using the pyruvate dehydrogenase complex (PDH). Acetyl CoA is the fuel for the citric acid cycle.

The Pyruvate Dehydrogenase Complex

The pyruvate dehydrogenase complex (PDH) links glycolysis to the citric acid cycle.The PDH, a component of the mitochondrial matrix, is composed of three distinct enzymes that oxidatively decarboxylate pyruvate to form acetyl CoA. This reaction is an irreversible link between glycolysis and the citric acid cycle

Coenzyme A

Coenzyme A (CoA) is a carrier of acyl groups derived from the vitamin pantothenate.Acyl groups (such as the acetyl group) are important constituents both in catabolism &anabolism. The transfer of the acyl group is highly exergonic because the thioester is unstable

Overview of Cellular Respiration

The citric acid cycle removes electrons from acetyl CoA and uses these electrons to reduce NAD + and FAD to form NADH and FADH 2. The electron-transport chain is a series of membrane proteins that electrons released in the reoxidation of NADH andFADH 2 flow through to generate a proton gradient across the inner mitochondrial membrane. Protons flow through ATP synthase to generate ATP from ADP and inorganic phosphate. The Electron-transport chain and ATP synthase compose oxidative phosphorylation.

List the three enzymes and five coenzymes that make up the pyruvate dehydrogenase complex and describe each of their roles

the pyruvate dehydrogenase complex is a large, highly integrated complex of three distinct enzymes. The enzymes are pyruvate dehydrogenase component (E1),dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3).

coenzymes:

thiamine pyrophosphate

lipoic acid

coenzyme A

flavin adenine dinucleotide (FAD)

nicotinamide adenine dinucleotide (NAD+)

thiamine pyrophosphate

decarboxylates pyruvate, yielding hydroxyethyl

lipoic acid

accepts hydroxyethyl carbanion from TTP as acetyl group

coenzyme A

accepts acetyl group from lipoamide

flavin adenine dinucleotide (FAD)

accepts pair of electrons from reduced lipoamide

nicotinamide adenine dinucleotide (NAD+)

accepts a pair of electrons from reduced FADH2

Differentiate between catalytic and stoichiometric coenzymes. Know what each coenzyme is an activated carrier for (i.e., what group is carried?)

The coenzymes thiamine pyrophosphate (TPP), lipoic acid, and FAD are catalyticcoenzymes, whereas CoA and NAD + are stochiometric coenzymes (i.e., coenzymesthat function as substrates). TPP is an activated carrier of aldehyde, lipoic acid and CoA are activated carriers of acyl groups, and FAD and NAD+ are activated carriers of electrons

describe the mechanistic steps in the pyruvate dehydrogenase complex

Step 1: Decarboxylation

Step 2: Oxidation and transfer to lipoamide

Step 3: Formation of Acetyl CoA

Step 4: Regeneration of oxidized lipoamide

Step 5: Regeneration of oxidized E3

Step 1: Decarboxylation

Pyruvate combines with TPP and is then decarboxylated to yield hydroxyethyl-TPP. Pyruvate dehydrogenase (E 1 ), a component of the complex, catalyzes the decarboxylation. Pyruvate combines with the ionized form of the coenzyme thiamine pyrophosphate (TPP).

Step 2: Oxidation and transfer to lipoamide

the hydroxyethyl group attached to TPP is oxidized to form an acetyl group while being simultaneously transferred to lipoamide, a derivative of lipoic acid that is linked to the side chain of a lysine residue by an amide linkage. The oxidant in this reaction is the disulfide group of lipoamide, which is reduced to its disulfhydryl form. This reaction, also catalyzed by the pyruvate dehydrogenase component E1, yields acetyl lipoamide.

Step 3: Formation of Acetyl CoA

The acetyl group is transferred from acetyl lipoamide to CoA to form acetyl CoA. Dihydrolipoyl transacetylase (E2) catalyzes this reaction. The energy-rich thioester bond is preserved as the acetyl group is transferred to CoA. Acetyl CoA, the fuel for the citric acid cycle, has now been generated from pyruvate

Step 4: Regeneration of oxidized lipoamide

To participate in another reaction cycle, dihydrolipoamide must be reoxidized. This reaction is catalyzed by dihydrolipoyl dehydrogenase (E 3). E3 uses a disulfide bond and an FAD cofactor. Lipoamide is oxidized by E3's disulfide

Step 5: Regeneration of oxidized E3

To participate in another reaction cycle, E3 itself must be reoxidized. This reaction isalso catalyzed by dihydrolipoyl dehydrogenase (E 3). Electrons move to FAD to form FADH 2. Electrons move from FADH 2 to NAD + to form NADH and H +. E3 is restored to its oxidized state

The Pyruvate Dehydrogenase Complex Is Tightly Regulated - Why?

The formation of acetyl CoA from pyruvate is irreversible in animal cells. Acetyl CoAhas two principal fates: metabolism by the citric acid cycle or incorporation into fatty acids.

Most pyruvate is derived from glucose by glycolysis. Therefore, we only want pyruvate to be converted to acetylCoA when two conditions are met:

1. Low NADH

2. Low Acetyl CoA.

This spares glucose since pyruvate can be used to make glucose (gluconeogenesis) but acetyl CoA cannot. This means that NADH and Acetyl CoA are potent inhibitors of the pyruvate dehydrogenase complex

The Pyruvate Dehydrogenase Complex Is Tightly Regulated - How?

The pyruvate dehydrogenase complex is regulated in two key modes - 1) feedbackinhibition and 2) reversible covalent modification (phosphorylation & dephosphorylation)

In either case (NADH & acetyl CoA),there is no need to metabolize pyruvateto acetyl CoA.• This inhibition has the effect of sparing glucose, because most pyruvate is derived from glucose by glycolysis

Phosphorylation/Dephosphorylation of the Pyruvate Dehydrogenase Complex

Enzyme E 1 is a key site of regulation. A kinase associated with the complex (pyruvate dehydrogenase kinase, PDH kinase) phosphorylates and inactivates E 1. A phosphatase (pyruvate dehydrogenase phosphatase, PDH phosphatase), also associated with the complex, removes the phosphate and thereby activates the enzyme.Both the kinase and the phosphatase are regulated.

pyruvate dehydrogenase phosphatase deficiency

Individuals with pyruvate dehydrogenase phosphatase deficiency have a pyruvate dehydrogenase complex that is always phosphorylated (i.e.,inactive)

.• In these individuals, glucose is processed to lactate rather than to acetyl CoA, and high blood lactic acid results.

• Many systems malfunction in the acidified environment, particularly the central nervous system.

• Treatment: dichloroacetate (DCA)and ketogenic diet

diabetic neuropathy

Diabetic neuropathy may be due to inhibition of the pyruvate dehydrogenase complex

Experimental treatments:

1) Inhibition of either PDH kinase or LDH enzyme

2) Gene editing to remove the PDH kinase gene

beriberi

The disruption of pyruvate metabolism is the cause of beriberi

Pyruvate dehydrogenase (E 1 ) catalyzes the decarboxylation of pyruvate by combining pyruvate with the ionized form of the coenzyme thiamine pyrophosphate (TPP)

mercury & arsenic poisoning

Poisoning by mercury and arsenic result in disruption of pyruvate metabolism

Explain how enhanced PDH Kinase activity facilitates the development of cancer by giving a rationale about how this process promotes aerobic glycolysis (the Warburg effect).

by suppressing mitochondrial glucose oxidation, redirecting the flow of glucose metabolites toward aerobic glycolysis. This metabolic shift supports rapid cancer cell proliferation and survival by providing the necessary building blocks for growth while reducing cell-damaging reactive oxygen species