Introduction
Pyruvate, the simplest of the α-keto acids, and other α-keto acids play pivotal roles in cellular metabolism, acting as key intermediates that connect various metabolic pathways. These compounds are not only essential for energy production but also serve as precursors for the synthesis of other vital biomolecules. This article explores the multifaceted roles of pyruvate and α-keto acids, from their involvement in core metabolic processes to their emerging functions in cellular signaling and disease development.
What are α-Keto Acids?
An α-keto acid is an organic compound characterized by a ketone group (C=O) adjacent to a carboxyl group (COOH). Pyruvic acid (CH3COCOOH) is the simplest example. These acids are crucial intermediates in the catabolism of lipids and proteins, playing a vital role in energy production and metabolism.
Pyruvate: A Central Metabolic Intermediate
Pyruvate is a key chemical compound in biochemistry, serving as the output of glucose metabolism via glycolysis. One molecule of glucose breaks down into two molecules of pyruvate. Pyruvate stands at a critical intersection in the network of metabolic pathways, with several potential fates:
- Conversion to Acetyl-CoA: Pyruvate can be converted into acetyl-coenzyme A (acetyl-CoA), which enters the Krebs cycle (also known as the citric acid cycle or tricarboxylic acid cycle) for further energy production. This process, called pyruvate decarboxylation, occurs inside the mitochondria after pyruvate is transported from the cytosol.
- Anaerobic Breakdown: In the absence of sufficient oxygen, pyruvate undergoes anaerobic breakdown, producing lactate in animals and ethanol in plants and microorganisms.
- Gluconeogenesis: Pyruvate can be converted into carbohydrates via gluconeogenesis.
- Fatty Acid Synthesis: Pyruvate can be converted to fatty acids or energy through acetyl-CoA.
- Amino Acid Synthesis: Pyruvate can be converted to the amino acid alanine.
In the last step of glycolysis, phosphoenolpyruvate (PEP) is converted to pyruvate by pyruvate kinase.
The α-Ketoacid Dehydrogenase Complexes (KDHc)
The α-keto acid dehydrogenase complex (KDHc) class of mitochondrial enzymes is composed of four members: pyruvate dehydrogenase (PDHc), α-ketoglutarate dehydrogenase (KGDHc), branched-chain keto acid dehydrogenase (BCKDHc), and 2-oxoadipate dehydrogenase (OADHc). These enzyme complexes occupy critical metabolic intersections that connect monosaccharide, amino acid, and fatty acid metabolism to Krebs cycle flux and oxidative phosphorylation (OxPhos). This feature also imbues KDHc enzymes with the heightened capacity to serve as platforms for propagation of intracellular and intercellular signaling.
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The KDHc enzymes also play important roles in the modulation of mitochondrial metabolism and epigenetic programming in the nucleus through the provision of various acyl-CoAs, which are used to acylate proteinaceous lysine residues. Intriguingly, nucleosomal control by acylation is also achieved through PDHc and KGDHc localization to the nuclear lumen.
Common Features of KDHc Enzymes
Although each KDHc fulfills a distinct catabolic role, the four dehydrogenases in this family of enzymes share common features in terms of their structure, overall composition, catalytic mechanism, regulation by allosteric factors, covalent modifications, and control by ions and the products they form. The oxidative decarboxylation of KDHc substrates, pyruvate (PDHc), α-ketoglutarate (KGDHc), branched-chain keto acids (BCKA; BCKDHc), and 2-oxoadipate (OADHc), respectively, results in the formation of acyl-CoAs and NADH. The acyl-CoAs are oxidized further by the Krebs cycle and NADH produced by the KDHc injects electrons into the electron transport chain (ETC) through complex I to drive OxPhos.
Individual KDHc Enzymes and Their Roles
Pyruvate Dehydrogenase Complex (PDHc)
PDHc connects glycolysis to the Krebs cycle by converting pyruvate formed by monosaccharide metabolism into acetyl-CoA. The acetyl-CoA is then converted to citrate through a Claisen-type condensation reaction catalyzed by citrate synthase in the presence of oxaloacetate. The amino acids Ala, Gly, Cys, Ser, and Thr also form pyruvate through transamination reactions, making PDH an important point for the intake of certain amino acids into the Krebs cycle. PDHc is heavily regulated by a kinase (PDHK) and phosphatase (PDP). Cells contain several PDK isoforms (PDK 1-4) which are expressed in specific tissues. PDK1-4 inhibits PDHc in response to hypoxia, nutrient deprivation, and calorie restriction, and when fatty acid (FAO) rates are high. Notably, defects in PDK function has been implicated in the progression of several diseases like diabetic cardiomyopathy, cancer, thrombosis, and cholestasis.
α-Ketoglutarate Dehydrogenase Complex (KGDHc)
KGDHc is the fourth enzyme in the Krebs cycle, and it catalyzes the oxidative degradation of α-ketoglutarate to succinyl-CoA. The succinyl-CoA is then metabolized further by the Krebs cycle, producing ATP through succinyl-CoA synthetase, which forms succinate, the substrate for succinate dehydrogenase (complex II) of the ETC. KGDHc is vital for amino acid metabolism because it is required for glutamate biosynthesis, the main source and sink for amines in cells. Glutamate catabolism is mediated by glutamate dehydrogenase (GDH), which couples its reversible oxidative deamination to the formation of α-ketoglutarate. Notably, glutamate is also used as an amide source for the biosynthesis of other amino acids. For example, glutamate is used to produce aspartate from oxaloacetate, a reaction catalyzed by aspartate aminotransferase (AAT). This forms α-ketoglutarate, which is metabolized by KGDHc. Thus, KGDHc activity is vital for amino acid homeostasis and the biosynthesis of cell proteins. Glutamate is also a neurotransmitter that modulates various neural functions and therefore KGDHc activity is integral for sustaining neuronal signaling. Defects in KGDHc can cause glutamate excitotoxicity, a type of cell death triggered by excessive glutamate production by neurons and glial cells. Glutamate is also an important fuel for cancer cell hyper-proliferation and survival. Cancer cells rewire glutamate/glutamine and Krebs cycle metabolism to promote tumorigenesis.
Branched-Chain Keto Acid Dehydrogenase Complex (BCKDHc)
BCKDHc is required for the degradation of branched chain amino acids (BCAA) leucine (Leu), isoleucine (Ile), and valine (Val). BCAA metabolism begins when branched chain amino acid transferase (BCAT) transaminates α-ketoglutarate to form the corresponding branched-chain keto acids (BCKA), ketoleucine, ketoisoleucine, and ketovaline. These BCAAs are then oxidized by BCKDHc generating acetyl-, propionyl-, and/or succinyl-CoA, respectively. Genetic defects in the E1β subunit of BCKDH causes a rare autosomal recessive disease called Maple Syrup Urine Disease (MSUD), which is characterized by deficiencies in BCAA degradation and the accumulation of BCKAs in the urine. BCAA catabolism is also an interorgan pathway that involves communication between hepatocytes and other tissues like skeletal muscle. The liver is a principal site for BCKA metabolism in mammals. However, BCAAs must first be converted to BCKAs in other tissues like muscle due to the absence of BCAT in hepatocytes. Recent studies have shown defects in liver BCKDHc activity in the liver shifts BCKA oxidation to the muscle and other organs, resulting in BCAA accumulation and the disruption of lipid metabolism. This leads to glucose intolerance, heart failure, and obesity. Importantly, BCAA accumulation due to defective BCKDHc results in the inhibition of pyruvate carrier in mitochondria and the suppression of gluconeogenesis in hepatocytes. This likely contributes to the development of metabolic and cardiovascular diseases listed above. Like PDHc, BCKDHc is modulated by its phosphorylation and dephosphorylation, reactions that are mediated by branched chain ketoacid dehydrogenase kinase (BDK) and phosphatase PPM1k (aka PP2Cm). BDK deactivates BCKDHc whereas PPM1k has the opposite effect. Notably, the accumulation of BCAAs is also a marker for tumorigenesis, which has been linked to the overstimulation of BDK.
Read also: The role of alpha-keto acids in metabolism.
2-Oxoadipate Dehydrogenase Complex (OADHc)
OADHc, also known as DHTKD1, is required for the mitochondrial degradation of lysine and converts 2-oxoadipate to glutaryl-CoA and NADH. The glutaryl-CoA is metabolized to crotonyl-CoA by glutaryl-CoA dehydrogenase (GCDH), which is then converted to acetyl-CoA. Genetic studies have linked mutation in the Dhtkd1 gene, which encodes the E1 subunit of OADH, to the development of several neurological disorders and the onset of a disease called alpha-ketoadipic aciduria. Impaired OADH is also linked to insulin resistance, cardiovascular disease risks, and Charcot-Marie-Tooth neuropathy. Like other acyl-CoAs, glutaryl-CoA is used for the “glutarylation” of proteinaceous lysine residues, which can be reversed by the NAD+-dependent deglutarylase activity of sirtuins like Sirt5 in mitochondria. Notably, reversible glutarylation has been linked to regulation of proteins in many tissues and has even been suggested to be neuroprotective. Furthermore, defects in protein glutarylation, either due to inborn errors in OADH activity and/or loss of Sirt5, has been linked to insulin resistance, cardiovascular diseases, and neurological disorders. Sirt5 is a well-known NAD+-dependent deacetylase. However, it also possesses desuccinylase and deglutarylase activity meaning it is required for the modulation of overall lysine acylation status.
Structure and Catalytic Cycle of KDHc Enzymes
The KDHc enzymes are composed of the same basic subunits: E1 (α-ketoacid decarboxylase; KDC), E2 (dihydrolipoyl acyltransferase; DLAT), and E3 (dihydrolipoamide dehydrogenase; DLDH). However, they are distinct from one another in terms of the number of copies and orientation of the three subunits that are used to form the multimer complexes. PDHc has a stoichiometry for the E1:E2:E3 subunits of 40:40:20, which forms a multisubunit holoenzyme that is ∼9.5 MDa in size. By contrast, KGDH is predicted to contain ∼12 E1 and ∼12 E3 subunits surrounding a 24-mer E2 core, forming a multisubunit complex that is ∼3.2 MDa. BCKDHc contains ∼6-12 copies of the E1 and E3 subunits organized around a 24-meric cubic E2 core. OADHc is also comprised of multiple copies of the E1, E2, and E3 subunits but it is unclear how many of the three subunits are required to form the multi-subunit holoenzyme. Another distinguishing feature of the KDHc enzymes is only PDHc and BCKDHc are modulated by kinases and phosphatases. The kinases and phosphatases that are used by mammalian cells to deactivate and activate, respectively, PDHc and BCKDHc were briefly described above and were discussed in detail in several recent reviews. KGDH harbors an adaptor protein called KGD4 that was reported to be required for the assembly of the subunits into a functional complex. KGDHc is targeted for phosphorylation in Corynebacterium glutamicum by serine/threonine protein kinase G (PknG). However, there is currently no evidence showing mammalian KGDHc is controlled by reversible phosphorylation.
A variety of co-factors and prosthetic groups are utilized by KDHc to catalyze the multistep conversion of the α-keto acid to acyl-CoA and NADH. These cofactors and prosthetic groups are thiamine pyrophosphate (TPP), lipoic acid, CoASH, and NAD+. The conversion of α-keto acid to acyl-CoA and NADH is initiated by the decarboxylase activity of the E1 subunit. Here, the α-keto acid is bound and used to covalently modify the TPP. This drives the oxidative decarboxylation of the α-keto acid, forming an acylated TPP intermediate. The acyl group is then transferred from the E1 subunit to the oxidized lipoic acid on the E2 subunit, creating a thioester linkage between the acyl group and the vicinal thiols of the lipoate. The acyltransferase activity of the E2 subunit then facilitates a thiol disulfide exchange reaction between the acyl-lipoic acid and CoASH. This reduces the vicinal sulphydryl groups in lipoic acid, releasing acyl-CoA. The reducing equivalents in the dihydrolipoamide are then transferred to the tightly bound FAD + group located in the E3 subunit. This forms FADH2, which is then used by the E3 subunit to reduce NAD+ to NADH.
α-Keto Acids in Disease
Deficiencies or imbalances in α-keto acid metabolism can lead to various metabolic disorders. For example, a deficiency in the enzyme responsible for the oxidative decarboxylation of α-ketoglutarate can lead to maple syrup urine disease, characterized by the buildup of branched-chain α-keto acids and associated neurological and developmental problems. Similarly, an imbalance in the metabolism of homocysteine, an α-keto acid intermediate, can contribute to the development of homocystinuria, which is linked to an increased risk of cardiovascular and neurological complications. Defects in KDHc function can cause disease states through the disruption of cell redox homeodynamics and the deregulation of metabolic signaling.
Pyruvate as an Immunonutrient
Emerging evidence suggests that pyruvic acid may have immunonutritional significance in modulating host defense mechanisms and immunoregulation. This is due to its role in cellular energetics and metabolism, serving as a source of respiratory cellular fuel and metabolic precursors.
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Pyruvate Supplementation
Pyruvate is sometimes sold as a weight-loss supplement. However, the scientific evidence supporting this claim is limited. A systematic review of six trials found a statistically significant difference in body weight with pyruvate compared to placebo, but the trials had methodological weaknesses and the effect size was small. Adverse events associated with pyruvate supplementation include diarrhea, bloating, gas, and increased LDL cholesterol.