Glycolysis

Unit 4: Carb & Lipid Metabolism

Introduction

  • Course Title: Biochemistry 555

  • Instructor: Elizabeth Hull, PhD

  • Recommended Reading:

    • Marks ch 21

    • Marks ch 22

    • Lippincott ch 8

    • Glycolysis Review Video 1

Learning Objectives

  • Identify and articulate the differences in structure and digestion of:

    • Amylose (unbranched)

    • Amylopectin

    • Glycogen

    • Isomaltose

    • Maltotriose

    • Maltose

    • Compare with cellulose structure.

  • Understand the roles of brush border enzymes, disaccharidases, and transporters (GLUT2 and SGLT) in sugar absorption, especially for fructose and galactose, and their processing into glucose pathways.

  • Describe glycolysis, focusing on:

    • Input/output

    • Priming steps

    • Energy-producing steps

    • Net energy yield under aerobic and anaerobic conditions.

  • Explain the significance of G6P as a common metabolic intermediate and its connection to the rate-limiting regulatory step of glycolysis.

  • Analyze regulatory mechanisms of glycolysis, including enzyme names for:

    • Step 1 (Glucokinase, Hexokinase)

    • Step 3 (PFK-1)

    • Step 10 (Pyruvate Kinase)

  • Detail the actions and regulation of F2,6bisP concerning insulin and glucagon ratios.

  • Explore PK deficiency, its relation to mutant enzyme activity, anemia symptoms, and lack of symptoms in other tissues.

  • Discuss disease causation due to metabolic blocks and effects, including lactic acidosis.

Overview of Carbohydrate Metabolism

  • General Information:

    • Carbohydrates are fundamental dietary sources, constituting the largest caloric intake for the global population.

    • Acceptable Macronutrient Distribution Range (AMDR): 45-65% of total calories from carbohydrates.

Sources of Carbohydrates

  • Plant Sources:

    • Amylopectin

    • Amylose

    • Sucrose

    • Fiber (no calories, but provides nutritional benefits)

  • Animal Sources:

    • Glycogen

    • Glycolipids

    • Lactose

  • Glucose can be synthesized from amino acids when dietary glucose is deficient.

Carbohydrate Utilization in the Body

In the Fed State

  • Processes Involved:

    • Glycolysis: Converts glucose to Acetyl-CoA for the TCA cycle and Electron Transport Chain (ETC) to produce ATP.

    • Glycogenesis: Stores excess glucose as glycogen when energy needs are met.

    • HMP Shunt: Generates NADPH for anabolic processes.

In the Fasting State

  • Processes Involved:

    • Glycogenolysis: Decomposes stored glycogen to provide glucose for the TCA cycle.

    • Gluconeogenesis: Produces glucose from non-carbohydrate sources when glucose and glycogen are scarce.

Lecture Outline

  1. Glycolysis Overview: After digestion and absorption, glucose is converted to Acetyl-CoA and enters the TCA cycle and ETC for ATP production.

    • Stage 1: Digestion and Absorption of dietary carbohydrates, including enzymes involved.

    • Stage 2: Glycolysis, detailing the conversion of glucose to pyruvate and metabolism of monosaccharides, including regulatory mechanisms.

    • Stage 3: Energy production via ATP generation.

Stage 1: Digestion and Absorption

Dietary Carbohydrates

  • Monosaccharides: Glucose, Fructose, Galactose.

  • Disaccharides: Maltose, Lactose, Sucrose.

  • Polysaccharides:

    • Starch: Comes in two forms:

    • Amylose: Unbranched α1−4α1-4 polymer of glucose.

    • Amylopectin: branched α1−4α1-4 polymer with α1−6α1-6 branches approximately every 25 residues.

    • Glycogen: α1−4α1-4 polymer of glucose from animal sources with α1−6α1-6 branches every 15 residues.

    • Cellulose: Unbranched β1−4β1-4 glucose polymer from plants, indigestible for humans.

Digestion Process
Enzymatic Breakdown

  • Salivary α-amylase: Begins digestion in the mouth, cleaving α1−4α1-4 linkages but not α1−6α1-6 or β1−4β1-4 linkages.

  • Pancreatic α-amylase: Continues digestion in the small intestine after stomach acid neutralization, producing maltose, maltotriose, and isomaltose.

  • Brush Border Enzymes: Located in the intestinal mucosal membrane, responsible for breaking oligosaccharides and disaccharides down to monosaccharides:

    • Hydrolyases: α-dextrinase (cleaves α1−6α1-6) and glucoamylase (digests α1−4α1-4 in maltotriose).

    • Disaccharidases: Isomaltase, maltase, lactase, sucrase.

Carbohydrate Absorption

  • Monosaccharide Transporters:

    • GLUT Transporters: Assist in passive facilitated diffusion. Different GLUT subtypes have varying affinities (Km) and velocities (Vmax) to meet tissue needs.

    • GLUT2: High Km (low affinity) for intestinal glucose uptake.

    • SGLT Transporters: Active, Na+^+-dependent; specifically, SGLT1 transports glucose and galactose, making the energy cost justifiable for ATP production.

Stage 2: Glycolysis

Overview of Glycolysis

  • Goal: Converting dietary glucose to pyruvate for TCA cycle entry as acetyl-CoA.

  • Pathway Activity: Activated in the fed state by insulin and inhibited by glucagon.

  • Locations: Active in all tissues.

  • Mechanism: Comprised of 10 enzymatic reactions converting glucose to pyruvate:

    • The initial 5 reactions consume energy and split 6C glucose into two 3C glyceraldehyde-3-phosphate molecules.

    • The final 5 reactions produce energy and yield 2 pyruvate molecules.

Glycolysis Reaction Details
Inputs and Outputs

  • Input: 1 Glucose molecule, 2 NAD+^+.

  • Net Products: 2 Pyruvate molecules, 2 ATP, 2 NADH.

  • Energy Used:

    • Initial reactions consume 2 ATP (priming reactions).

    • One redox reaction stores electrons in 2 NADH.

    • Final reactions yield 4 ATP total (net gain of 2 ATP).

    • Under anaerobic conditions, pyruvate converts to lactate to regenerate NAD+^+, allowing continued glycolysis.

Regulatory Steps

  • 7/10 reactions are reversible with a ΔG° close to zero; 3 (steps 1, 3, 10) are irreversible, and involve large ΔG° shifts.

Glycolytic Pathway Steps

  1. Step 1: Priming reaction; ATP transfers phosphate to glucose, traps it in the cell.

  2. Step 2: Isomerization (aldose to ketose).

  3. Step 3: Second priming; ATP consumption.

  4. Step 4: Cleavage; produces two 3C molecules (dihydroxyacetone phosphate + glyceraldehyde-3-phosphate).

  5. Step 5: Isomerization of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate.

  6. Step 6: Glyceraldehyde-3-phosphate oxidation; energy stored in NADH and high-energy phosphate bond in 1,3-bisphosphoglycerate. Saturated phosphate bond via substrate-level phosphorylation.

    • Reaction has ΔG° = +1.5 kcal/mol.

  7. Step 7: High-energy phosphate transfer to ADP forming ATP; ΔG° = -4.5 kcal/mol, facilitating forward reaction.

    • Coupling of steps 6 and 7 leads to favorable conditions for glycolysis.

  8. Step 8: Moving phosphate from C3 to C2.

  9. Step 9: Removal of water; resonance stabilization of the phosphate bond.

  10. Step 10: Phosphorylation of ADP to ATP, producing pyruvate.

    • Reaction is energetically viable due to PEP's energy storage.

Metabolism of Other Monosaccharides

  • Galactose Metabolism:

    1. Phosphorylation to Galactose 1-P.

    2. Reaction with UDP-Glucose.

    3. UDP-Galactose converted to UDP-Glucose.

    4. Glucose 1-P enters various pathways as a common metabolic intermediate.

  • Fructose Metabolism (in the liver):

    1. Phosphorylated to Fructose 1-P.

    2. Aldolase B cleaves F1P generating glycolytic intermediates.

Glycolytic Regulation

Key Regulatory Steps

  1. Step 1 (Glucokinase Regulation):

    • Controlled by glucose concentration; activity is enhanced when glucose levels rise.

    • Feedback inhibition when F6P accumulates.

    • Hexokinase in other tissues, operates with feedback inhibition but has higher affinity to trap glucose in muscle.

  2. Step 3 (PFK-1 Regulation):

    • Allosterically controlled by ATP (negative) and AMP (positive); sensitive to citrate and fructose 2,6-bisphosphate for regulation of glycolytic flux.

  3. Step 10 (PK Regulation):

    • Phosphorylated and downregulated in fasting states via glucagon-induced signaling pathways.

    • Allosterically activated by fructose-1,6-bisphosphate.

Fructose 2,6-Bisphosphate Regulation

  • Acts as a potent positive allosteric effector of PFK-1.

  • Dephosphorylated form is active during high insulin and low glucagon states, amplifying glycolysis.

Stage 3: Energy Production

Energy Generating Mechanisms

  • ETC and Oxidative Phosphorylation:

    • Pyruvate derived from glycolysis enters the TCA cycle to generate ATP.

    • Yield is typically 30-32 ATP molecules generated.

Anaerobic Glycolysis Implications

  • Bypassed mitochondrial function during anaerobic conditions; net ATP yield under anaerobic conditions is 2 ATP per glucose.

  • Lactic acid production via lactate dehydrogenase regenerates NAD+.

    • Lactate buildup leads to lactic acidosis, indicative of metabolic blockage.

Other Functions of Glycolysis

  • Beyond ATP production, glycolytic pathways yield precursors for:

    • Nucleotide and sugar synthesis (ribose 5-P, UDP-glucose).

    • TAGs and amino acids.

    • Synthesis of 2,3-BPG, relevant for hemoglobin function in erythrocytes.

Metabolic Blocks

Definitions and Implications

  • Definition: Blocking one step in a metabolic pathway halts the entirety of the pathway.

  • Causes of Metabolic Blocks:

    • Genetic disorders leading to enzyme mutations.

    • Pharmacological interventions inhibiting enzyme activity.

    • Regulatory mechanisms triggered by allosteric effector build-up.

  • **Consequences of Metabolic Diseases:

    • Lack of product leading to symptomatic disease.

    • Toxicity arising from substrate accumulation upstream of blockage.

    • Potential re-routing into alternative pathways, creating complications.

Specific Example: Pyruvate Kinase and Hemolytic Anemia

  • RBCs lack mitochondria and rely solely on glycolysis, making them vulnerable to PK mutations.

  • PK mutation impacts ATP-generating steps, leading to reduced ATP, resulting in premature RBC lysis and anemia symptoms.

Key Concepts and Summary

  • Carbohydrates are primary acetyl-CoA sources for TCA cycle energy production in fed states; excess stored as glycogen.

  • Fasting State: Gluconeogenesis and glycogen breakdown to sustain glucose levels.

  • Glycolysis consists of 10 steps, with 5 dedicated to energy investment and 5 to generation, producing 2 ATP and 2 NADH.

  • Regulatory steps (1, 3, 10) crucial to maintaining metabolic homeostasis.

  • Lactic acidosis: Indicates a metabolic block due to anaerobic glycolysis or deficiencies in PDHC.

  • Mutations in associated enzymes can manifest as hematological conditions like hemolytic anemia, owing to disrupted ATP generation in red blood cells.