Section 8: The Citric Acid Cycle

CHAPTER 18: Preparation for the Cycle CHAPTER 19: Harvesting Electrons from the Cycle

Learning Objectives Explain why the reaction catalyzed by the pyruvate dehydrogenase complex is a crucial juncture in metabolism. Identify the means by which the pyruvate dehydrogenase complex is regulated. Identify the primary catabolic purpose of the citric acid cycle. Explain the advantage of the oxidation of acetyl CoA in the citric acid cycle. Describe how the citric acid cycle is regulated. Describe the role of the citric acid cycle in anabolism. Interactive Metabolic Map

Lehninger Principles of Biochemistry, 2017 Lecture: 11‐02‐2016

CHAPTER 18 Preparation for the Cycle Chapter 18 Outline Pyruvate dehydrogenase (PDH) is the one‐way link between glycolysis and cellular respiration, directing pyruvate into the formation of acetyl CoA

PDH Stages of catabolism (Chapter 15) Diverse fates of pyruvate (Chapter 16)

PDH LDH

ADH

Alcohol dehydrogenase (ADH); Lactate dehydrogenase (LDH); Pyruvate Dehydrogenase (PDH) Complex Under aerobic conditions, pyruvate enters the mitochondria where it is converted into acetyl CoA.

Acetyl CoA is the fuel for the citric acid cycle, which processes the two‐carbon acetyl unit to 2 molecules of CO2 while generating high‐ energy electrons that can be used to form ATP. An overview of the citric acid cycle

The citric acid cycle (CAC), The tricarboxylic acid (TCA) cycle or The Krebs Cycle; • Oxidizes two‐carbon units,

• Producing two molecules of CO2, • One molecule of ATP or GTP, • High‐transfer‐potential electrons.

• This is an aerobic process because it

requires oxygen (O2). This process is also called cellular respiration or oxidative phosphorylation.

• The CAC takes place in the mitochondria • Glycolysis takes place in the cytoplasm The pyruvate dehydrogenase complex, a mitochondrial matrix enzyme, oxidatively decarboxylates pyruvate to form acetyl CoA.

This reaction is an irreversible link between glycolysis and the citric acid cycle.

Mitochondrion The link between glycolysis and the citric acid cycle.

Cytoplasm Pyruvate produced by glycolysis is converted into acetyl CoA, the fuel of the citric acid cycle.

Mitochondria Fatty acid degradation is also an important source of acetyl CoA for the citric acid cycle (Chapter 27) The coenzymes thiamine pyrophosphate (TPP), lipoic acid, and flavin adenine dinucleotide (FAD) serve as catalytic coenzymes, and CoA and nicotinamide adenine dinucleotide (NAD+) are stoichiometric coenzymes. The synthesis of acetyl CoA from pyruvate consists of three steps: a decarboxylation, an oxidation, and the transfer to CoA.

This irreversible conversion of pyruvate into acetyl CoA is the link between glycolysis and the citric acid cycle Thiamine pyrophosphate is derived from the vitamin thiamine.

• Thiamine pyrophosphate coenzyme is the prosthetic group of three important enzymes: • Pyruvate dehydrogenase, • ‐ketoglutarate dehydrogenase (a citric acid cycle enzyme, Chapter 19) • Transketolase. Transketolase functions in the pentose phosphate pathway, which will be considered in (Chapter 26).

• The common feature of enzymatic reactions utilizing TPP is the transfer of an activated aldehyde unit. 1. Decarboxylation:

Pyruvate dehydrogenase (E1), a component of the complex, catalyzes the decarboxylation. Pyruvate combines with ionized form of the coenzyme thiamine pyrophosphate (TPP). 2. Oxidation:

The two‐carbon fragment is oxidized and transferred to dihydrolipoamide to form acetyllipoamide on E2 in a reaction also catalyzed by E1. Dihydrolipoamide is formed by the attachment of the vitamin lipoic acid to a lysine residue in another enzyme in the complex, dihydrolipoyl transacetylase (E2). 3. Formation of acetyl CoA:

E2 catalyzes the transfer of the acetyl group from acetyllipoamide to coenzyme A to from acetyl CoA.

To participate in another reaction cycle, dihydrolipoamide must be reoxidized.

This reaction is catalyzed by dihydrolipoamide dehydrogenase (E3). • The three enzymes of the pyruvate dehydrogenase complex are structurally integrated.

• The lipoamide arm allows rapid movement of substrates and products from one active site of the complex to another. Reactions of the pyruvate dehydrogenase complex. • The formation of acetyl CoA from pyruvate is irreversible in animal cells.

• Acetyl CoA has two principle fates: metabolism by the citric acid cycle or incorporation into fatty acids. Regulation of Pyruvate Dehydrogenase Complex

• Enzyme E1 is a key site of regulation. A kinase associated with the complex phosphorylates and inactivates E1.

• A phosphatase, also associated with the complex, removes the phosphate and thereby activates the enzyme.

• The pyruvate dehydrogenase complex is also regulated by energy charge.

• ATP, acetyl CoA, and NADH inhibit the complex.

• ADP and pyruvate stimulate the complex. Regulation of the pyruvate dehydrogenase complex by the energy charge Answer: The reaction is facilitated by having the active sites in proximity, which increases the overall reaction rate and minimizes side reactions. • Enhanced PDH kinase activity inhibits the pyruvate dehydrogenase complex.

• Pyruvate is subsequently processed to lactate, which can result in lactic acidosis.

• Production of lactate in the presence of oxygen is a characteristic of cancer cells.

Hypoxia inducible factor‐1, the transcription factor that enhances aerobic glycolysis in cancer cells, also stimulates the expression of pyruvate dehydrogenase kinase.

The subsequent inhibition of the dehydrogenase prevents the formation of acetyl CoA and directs pyruvate to the synthesis of lactic acid. • Thiamine deficiency results in insufficient pyruvate dehydrogenase activity.

• Insufficient pyruvate dehydrogenase activity causes neuromuscular pathologies, such as beriberi.

• The vitamin thiamine is found in brown rice, but not in white (polished) rice. Milled and polished rice Arsenite poisoning

Pyruvate dehydrogenase complex activity can be inhibited by mercury and arsenite, which bind to the two sulfurs of dihydrolipoamide.

2,3‐Dimercaptopropanol can counter the effects of arsenite poisoning by forming a complex with the arsenite that can be excreted.

Early hatters used mercury to make felt, which inhibited complex activity in the brain, often leading to strange behavior. Mad Hatter The Mad Hatter is one of the characters that Alice meets at a tea party in her journey through Wonderland.

Real hatters worked with mercury, which inhibited an enzyme responsible for providing the brain with energy.

The lack of energy would lead to peculiar behavior, often described as “mad.”