Glycolysis and Pyruvate Oxidation

• These study notes cover glycolysis, pyruvate oxidation, and related metabolic concepts.

Five Perspectives for Learning Metabolism

• Intermediary metabolism involves interacting pathways that extract/store energy from fuel molecules.
• Learning metabolism can be consistently organized using five perspectives.
• Pathway reaction steps: Each reaction has unique substrates, products, enzymes, cofactors, and inhibitors.
• Regulated reactions: Hormones and/or metabolites regulate certain steps to control metabolite flow.
• Unique characteristics: Each pathway has unique functional aspects and contributions to metabolism.
• Interface with other pathways: Metabolic intermediates serve as substrates for alternate pathways, linking pathways.
• Related diseases: Enzyme deficiencies disrupt metabolite availability, leading to homeostasis imbalances.

Pathway Reaction Steps

Glycolysis - Glucose to Pyruvate

• Glycolysis converts glucose to pyruvate in two stages:
• Requires energy to convert glucose to triose phosphates (5 reactions), produces energy by converting triose phosphates to pyruvate (5 reactions).

Conversion of Glucose to Glyceraldehyde 3-Phosphate

• Glucose is phosphorylated by hexokinase (or glucokinase in the liver) using ATP to trap glucose inside the cell. This step is irreversible.
• Glucose 6-phosphate (G6P) is converted to fructose 6-phosphate (F6P) by phosphoglucose isomerase, moving the carbonyl group.
• F6P acquires another phosphate from ATP via phosphofructokinase, producing fructose 1,6-bisphosphate (F1,6-BP).
• F1,6-BP is cleaved by aldolase into dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P).
• DHAP is isomerized to G3P by triose phosphate isomerase, allowing two G3P molecules to form from one F1,6-BP.

Conversion of Glyceraldehyde 3-Phosphate to Pyruvate

• G3P is oxidized and phosphorylated by glyceraldehyde 3-phosphate dehydrogenase, producing 1,3-bisphosphoglycerate (1,3-BPG) and NADH using inorganic phosphate.
• A phosphate is transferred from 1,3-BPG to ADP by phosphoglycerate kinase, producing ATP and 3-phosphoglycerate (3PG). This is substrate-level phosphorylation.
• The phosphate group on 3PG is shifted to carbon 2 by phosphoglyceromutase to produce 2-phosphoglycerate (2PG).
• A molecule of water is removed from 2PG by enolase, producing phosphoenolpyruvate (PEP.)
• Fluoride inhibits enolase by binding with Mg++.
• PEP undergoes substrate-level phosphorylation of ADP via pyruvate kinase, yielding ATP and pyruvate. This is another substrate-level phosphorylation.

Pyruvate Oxidation - Pyruvate to Acetyl-Coenzyme A

• Pyruvate is transported into the mitochondrial matrix and oxidized by the pyruvate dehydrogenase complex (PDC), linking glycolysis to the citric acid cycle.
• Three steps produce acetyl-CoA and NADH.
• Pyruvate binds to thiamine pyrophosphate (TPP) on the pyruvate dehydrogenase (PDH) enzyme and is decarboxylated, releasing CO2.
• A hydroxyethyl group, with two carbons from pyruvate, remains bound to TPP.
• The hydroxyethyl group is transferred from TPP to lipoic acid by dihydrolipoyl transacetylase; lipoic acid is reduced, and the hydroxyethyl group is oxidized to an acetyl group.
• The acetyl group is transferred to CoA, producing acetyl-CoA, leaving lipoyl coenzyme in the reduced form.
• The reduced lipoyl coenzyme is oxidized by dihydrolipoyl dehydrogenase, using FAD as a coenzyme, which is reduced to FADH2. FADH2 reduces NAD+ to NADH.

Regulated Reactions

Regulation of Glycolysis

• Glycolysis is regulated at three points, each with a different function.
• Hexokinase (except in the liver) is allosterically inhibited by G6P, ensuring cells don't take up too much glucose, while glucokinase in the liver is not inhibited by G6P.
• Phosphofructokinase (PFK-1) controls G6P entry into glycolysis; when slowed, G6P is routed to glycogen synthesis/pentose phosphate pathway.
• Fructose 2,6-bisphosphate (F2,6-BP) stimulates PFK-1 in the liver when insulin levels are high. Elevated glucagon inhibits PFK-2, lowering F2,6-BP.
• Adenosine monophosphate (AMP) stimulates PFK-1 in muscle during exercise to restore ATP levels.
• ATP and citrate slow glycolysis when energy is abundant.
• Pyruvate kinase regulation controls PEP flow to pyruvate/gluconeogenesis.
• Pyruvate kinase is allosterically stimulated by F1,6-BP in well-fed conditions and inhibited by ATP and alanine during fasting.

Regulation of Pyruvate Oxidation

• The PDC is regulated by covalent modification of pyruvate dehydrogenase (PDH).
• PDH kinase inactivates PDH by phosphorylation with ATP. Reactivation occurs via PDH phosphatase.
• PDH kinase is stimulated by NADH and acetyl-CoA and is inhibited by pyruvate.
• PDH phosphatase is stimulated by Ca++ and insulin.

Unique Characteristics

Anaerobic Glycolysis

• The capacity to recycle NADH back to NAD+ anaerobically is important in several tissues.

Liver

• The conversion of pyruvate to lactate recycles NADH, allowing the liver to dispose of excess NADH or pyruvate.
• Lactate can be converted back to pyruvate or excreted.
• The net energy production of anaerobic glycolysis is 2 ATP per glucose molecule; no CO2 is produced.

Muscle

• Fast-twitch muscle fibers use glycolysis for rapid energy, containing high concentrations of lactate dehydrogenase.

Red Blood Cells

• Red blood cells (RBCs) lack mitochondria, relying entirely on anaerobic metabolism for energy.

Glucokinase Versus Hexokinase

• Glucokinase, the isoform in the liver, captures dietary glucose; its high Km minimizes glucose uptake during fasting, preventing hypoglycemia.
• Glucokinase is also present in pancreatic B cells.
• Hexokinase, the widely distributed isoform, has a low Km, allowing glucose entry into brain cells and RBCs under fasting.

Cellular Compartmentation

• Glycolysis occurs in the cytoplasm; the PDH pathway is in the mitochondrial matrix.
• Cells that rely on anaerobic glycolysis, like fast-twitch muscle fibers and RBCs, have few mitochondria.

Function of Glucokinase in B cells

• B cells contain glucokinase instead of hexokinase to prevent inappropriate insulin secretion. Elevated G6P signals insulin release when blood glucose rises.

Multienzyme Complexes

• The PDC exemplifies a multienzyme unit with coordinated function via geometric arrangement, preventing intermediate diffusion.

Energy Production

• ATP yield from glucose oxidation depends on O2 availability.
• Under aerobic conditions, complete conversion yields 36-38 ATP/glucose.
• Under anaerobic conditions, conversion yields 2 ATP/glucose.

Interface with Other Pathways

Glucose 6-Phosphate

• Because glucose 6-phosphate is also a product of gluconeogenesis, it serves as a substrate for glucose-6-phosphatase in the liver. The action of this enzyme releases free glucose into the bloodstream.
• Phosphoglucomutase provides for interchange between glycogen, galactose, and uronic acid metabolism.

Fructose 6-Phosphate

• F6P is the precursor for amino sugar synthesis.

Dihydroxyacetone Phosphate

• DHAP is converted to glycerol 3-phosphate, providing a source for triglyceride and phospholipid metabolism and carbons for gluconeogenesis.

Pyruvate

• Pyruvate is converted to oxaloacetate by pyruvate carboxylase for gluconeogenesis.
• Pyruvate is interconverted with alanine by alanine aminotransferase (alanine cycle) and with lactate (Cori cycle).

Acetyl-Coenzyme A

• Acetyl-CoA is a precursor for fatty acid synthesis and product of fatty acid oxidation, ethanol catabolism, and ketone body catabolism.

Related Diseases

Lactic Acidosis

• Lactic acidosis results from increased lactate in the blood:
• This is typically due to overproduction in the liver or skeletal muscle.
• Increased NADH (from hypoxia, respiratory distress, shock, or ethanol consumption) or increased pyruvate (from PDH deficiency or pyruvate carboxylase deficiency) can lead to lactic acidosis.

Pyruvate Kinase Deficiency

• Pyruvate kinase deficiency is the most common enzyme deficiency in glycolysis, severely reducing ATP production in RBCs, leading to hemolytic anemia.

Pyruvate Dehydrogenase Deficiency

• Deficiencies in PDC components increase lactate and cause lactic acidosis and reduced citric acid cycle activity, leading to myopathy and neuropathy.

Arsenate and Arsenite Poisoning

• Arsenate uncouples substrate-level phosphorylation by G3P dehydrogenase, eliminating ATP gain from anaerobic glycolysis.

Arsenite Poisoning

• Arsenite binds covalently to lipoic acid, preventing hydroxyethyl group transfer.