Principles of Biochemistry, Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
The Citric Acid Cycle
Pyruvate must first be oxidized to acetyl-CoA and CO2

CARBOHYDRATES, Fatty acids, and Most Amino Acids are ultimately oxidized through The Citric Acid Cycle to CO₂ and H₂O. However, before these nutrients can enter the cycle, their carbon skeletons must be broken down and their fragments converted into the acetyl groups of acetyl-CoA, as it is in this form that The Citric Acid cycle accepts the bulk of its incoming fuel. Chapters 18 and 19 detail how acetyl groups are formed from Fatty Acids and amino acids for this cycle. Here, we examine the process by which Pyruvate, generated from the hydrolytic breakdown of glucose, is oxidized to acetyl-CoA and CO₂ through the action of a set of Enzymes structurally organized into the pyruvate dehydrogenase complex. This multienzyme system, located in the Cell/35.html">Mitochondria of Eukaryotic Cells and in the Cytoplasm of prokaryotes, catalyzes the following overall reaction:

Class="center">Pyruvate + NAD⁺ + CoA-SH →

→ Acetyl-CoA + NADH + СО₂

∆G⁰' = -8.0 kcal/mol.

This rather complex reaction involves The oxidative decarboxylation of pyruvate—a dehydrogenation process in which the carboxyl group of pyruvate is removed as a molecule of CO₂, and its acetyl group is incorporated into acetyl-CoA. One hydrogen atom split off from pyruvate appears in NADH, and the other as H⁺. The resulting NADH then transfers its electrons to the Electron Transport Chain (Fig. 16-1), through which they are ultimately delivered to molecular oxygen.

The combined dehydrogenation and decarboxylation of pyruvate to acetyl-CoA involves three successive enzymes: pyruvate dehydrogenase (E₁), dihydrolipoyl transacetylase (E₂), and dihydrolipoyl dehydrogenase (E₃), along with five Coenzymes or prosthetic groups—thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA), nicotinamide adenine dinucleotide (NAD⁺), and Lipoic Acid. All of these Enzymes and Coenzymes are assembled into a multienzyme system, which was first isolated and thoroughly studied by Lester Reed and his colleagues at the University of Texas. Four of the Vitamins required by The Human Body are essential components of this very system: thiamine (in TPP), riboflavin (in FAD), pantothenic acid (in CoA), and nicotinamide (in NAD⁺). In addition, the reaction requires lipoic acid (Fig. 16-3); it acts as an essential vitamin or growth factor in certain microorganisms, whereas higher animals are able to synthesize it from readily available precursors. The pyruvate dehydrogenase complex isolated from E. coli cells is a particle approximately 45 nm in diameter, making it somewhat larger than a ribosome. The Molecular Weight of this large multienzyme system exceeds 6 ∙ 10⁶. At the center of the complex, forming a "core" to which the Other Enzymes are attached, lies dihydrolipoyl transacetylase (molecular weight 200,000). Its molecule consists of 24 subunits—polypeptide chains, in each of which two lipoyl groups are attached to two specific Lysine residues in the active center of the subunit (attachment occurs via The formation of a peptide bond between the carboxyl group of lipoic acid and the ε-amino group of the lysine residue; Fig. 16-3). Attached to the dihydrolipoyl transacetylase are very large molecules of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase. Pyruvate dehydrogenase contains bound thiamine pyrophosphate, and dihydrolipoyl dehydrogenase contains bound FAD.

Fig. 16-3. Lipoic acid and its active form, which serves as the prosthetic group of dihydrolipoyl transacetylase. Lipoic acid and the lipoyl group can exist in oxidized (disulfide), reduced (dithiol), and acetylated forms. Therefore, the lipoyl group acts as both a hydrogen carrier and an acetyl group carrier. The lipoyl-lysine arm, about 1.4 nm long, acts as a "swiveling arm." It transfers hydrogen atoms from pyruvate dehydrogenase to dihydrolipoyl dehydrogenase, as well as the acetyl group to CoA-SH. Lipoic acid is sometimes referred to as a pseudovitamin.

The lipoyl-lysine groups of the central enzyme in the pyruvate dehydrogenase complex, measuring about 1.4 nm in length, function as "swiveling arms" that transfer hydrogen atoms and acetyl groups from one enzyme molecule of the complex to another. Two other enzymes that regulate the pyruvate dehydrogenase reaction are also attached to the pyruvate dehydrogenase complex (discussed below).

Figure 16-4 schematically illustrates The sequence of Reactions Catalyzed by the pyruvate dehydrogenase complex, through which the decarboxylation and dehydrogenation of pyruvate take place. This sequence comprises five stages. In stage 1, pyruvate loses its carboxyl group through interaction with thiamine pyrophosphate bound to pyruvate dehydrogenase (E₁); this reaction yields a hydroxyethyl derivative of thiamine pyrophosphate with a hydroxyethyl group at the thiazole ring (Section 10.4). Pyruvate dehydrogenase also catalyzes stage 2—The transfer of hydrogen atoms and the acetyl group from thiamine pyrophosphate to the oxidized form of the lipoyl-lysine prosthetic groups of the complex's central enzyme, dihydrolipoyl transacetylase, forming the 6-acetyl thioester of the reduced lipoyl groups. In Stage 3, a molecule of CoA-SH interacts with the acetyl derivative of dihydrolipoyl transacetylase, resulting in the Formation of Acetyl-S-CoA and the fully reduced, or dithiol, form of the lipoyl groups. In stage 4, this reduced form of dihydrolipoyl transacetylase is acted upon by dihydrolipoyl dehydrogenase, which catalyzes the transfer of hydrogen atoms from the reduced lipoyl groups to FAD, which acts as the prosthetic group of dihydrolipoyl dehydrogenase. In stage 5 (the final step in this reaction sequence), the reduced FAD group of dihydrolipoyl dehydrogenase transfers hydrogen to NAD⁺ to form NADH. The lipoyl-lysine side chains of dihydrolipoyl transacetylase play a crucial role in this process by transferring hydrogen atoms and the acetyl group from one enzyme to another. All these enzymes and coenzymes are structurally organized into a single complex, bringing the prosthetic groups into close proximity and allowing intermediates to interact rapidly with one another. If these very large enzyme molecules were dissociated and free to move in the Cytosol, they would have to travel considerable distances by diffusion before they could collide and interact. Figure 16-5 shows an electron micrograph of the pyruvate dehydrogenase complex.

Fig. 16-4 Stages of the Oxidative Decarboxylation of pyruvate to acetyl-CoA catalyzed by the pyruvate dehydrogenase complex. To make it easier to follow The Fate of pyruvate, its transformations are highlighted in red. The Structure of thiamine pyrophosphate and its α-hydroxyethyl derivative is shown in Fig. 10-2. E₁, pyruvate dehydrogenase; TPP, thiamine pyrophosphate; TPP-CHOH-CH₃, α-hydroxyethylthiamine pyrophosphate; E₂, dihydrolipoyl transacetylase; E₃, dihydrolipoyl dehydrogenase.

A deficiency of vitamin B₁, or thiamine (Section 10.4), causes the disease known as beriberi. It is now clear that in animals deprived of thiamine, the normal Oxidation of Pyruvate becomes impossible. This impairment particularly affects the Brain, which normally derives all its energy from the aerobic oxidation of glucose and for which pyruvate oxidation is therefore a vital process. The polyneuritis and general dysfunction of the motor Nervous system characteristic of beriberi (Chapter 10) are caused by impaired pyruvate dehydrogenase function.

Fig. 16-5. Electron micrograph of the pyruvate dehydrogenase complex isolated from E. coli cells. The particles of the complex are seen to be composed of subunits.

It is important to note that the process catalyzed by the pyruvate dehydrogenase complex in animal Tissues is irreversible. This has been confirmed by isotope experiments, which showed that radioactive CO₂ was not reincorporated into acetyl-CoA; that is, no carboxyl-labeled pyruvate was formed.

As we will see below, The regulation of pyruvate dehydrogenase complex activity constitutes a key element in the biological control of Respiration.