02: Carbohydrate Metabolism: Glycolysis and Gluconeogenesis

Glycolysis

Carbohydrate digestion

• begins in the mouth

• salivary amylase cleaves α- (1,4)-glycosidic bonds of ingested carbohydrates

  • Salivary amylase is inactivated by the stomach acid

  • Pancreatic secretions neutralize stomach acid

  • Oligosaccharides to smaller saccharides; disaccharides further broken down to monosaccharides

• Glucose is stored in the liver as glycogen or released in the bloodstream

  • sugars is taken up in portal circulation

• 

  • pancreatic a-amylase neutralizes and breaks down sugar

Glucose

• is polar because of OH groups —> promotes water solubility

• Hexose, monosaccharide

• The major form of carbohydrate presented to cells after absorption from

  food

• Glucose is what fuels the brain, and many other tissues

  • Brain weighs about 2% of body weight, but receives 20% of total body oxygen consumption, and 25% of total body glucose utilization

• Glucose is ‘burned’ to release energy

  • Biochemical pathways

• Uptake in cells is through transporter mechanisms

  • GLUT transporters

Glucose Transport

• Glucose cannot diffuse directly into the cells

• Na+ -independent facilitated diffusion

  • Glucose moves from region of high conc. to low conc.

  • Mediated by a family of Glucose Transporters (GLUT)

• Na+ -dependent co-transporter system

  • Transport of glucose from low glucose conc. (outside cell) to higher

    glucose conc

    • Process uses energy

  • Glucose transport follows the Na+ gradient into the cell

    • Sodium-dependent GLUTs aka SGLT

• GLUTs can transport glucose in two ways:

  • Extracellular to intracellular

    • Easier along the gradient

  • Intracellular to extracellular

• 

  • GLUT 1, 3, 6 transports glucose to the brain to ensure that brains always receives a supply of glucose

  • GLUT 5 transports fructose only (no glucose)

Glucose Transporters (GLUTs)

• Two conformational states

• Extra-cellular glucose molecule binds to the GLUT (conformational state 1)

• The transporter undergoes a change in its conformation (conformational state 2)

• The glucose molecule is transported across the cell membrane

• 

Glycolysis

• Primary pathway for the metabolism of glucose

  • Glycolysis = breakdown (lysis) of glucose

    • Sometimes, other carbohydrates are modified to glucose

• Location: all the cells of the body, in the cytosol

• Chemically, what is glycolysis?

  • It is a pathway

  • Series of chemical reactions where:

  • One molecule of glucose (precursor; 6 carbon atoms) is broken down (through many steps) to finally yield two molecules of pyruvic acid (final product, 3 carbon atoms)

  • Pyruvic acid aka pyruvate

    This results in the release of energy in the form of ATP

  • Catabolic process (anabolic, catabolic)

• Aerobic Glycolysis

  • Presence of oxygen

  • Important to oxidize NADH+ formed in glycolysis

    • Step 6: oxidation of glyceraldehyde-3-phosphate

  • Pyruvate converted to Acetyl CoA in the preparatory step to the Krebs cycle

    (TCA cycle, Citric Acid cycle)

• Anaerobic glycolysis

  • Absence of oxygen

  • Pyruvate is converted to lactate

    • Allows the production of energy in cells lacking mitochondria (erythrocytes) and cells deprived of oxygen

    • Cori cycle

  • Aka fermentation

Overview of Glycolysis

• Glycolytic pathway is employed by all tissues

• Series of 10 reactions

• Breakdown of glucose to provide energy (as ATP)

  • Also provides intermediates for other metabolic pathways

• Glycolysis is the hub of carbohydrate metabolism

  • All sugars (dietary, metabolic reactions and byproducts) can be converted to glucose

Investment phase of Glycolysis

• 

  Payoff Phase of Glycolysis

  • 

    • the payoff phase yields:

      • energy conserved as 2 ATP and 2 NADH

      • 2 pyruvate

Transition from Investment Phase to Payoff phase

• Fructose 1,6-bisphosphate is broken down into

  • Glyceraldehyde 3-phosphate and

  • Dihydroxyacetone phosphate

• 50%-50%

  • Both are C-3 compounds

• Aldotriose and Ketotriose – these are interconvertible

  • 

• Triose phosphate isomerase

  • Glyceraldehyde 3-phosphate remains intact

  • DHAP is converted to Glyceraldehyde 3-phosphate

• Now you have 100% Glyceraldehyde 3-phosphate

  • Taken into the payoff phase

Noteworthy Chemical Transformations of Glycolysis

• Three noteworthy chemical transformations:

  • 1) degradation of the carbon skeleton of glucose to yield pyruvate

  • 2) phosphorylation of ADP to ATP by compounds with high phosphoryl group transfer potential, formed during glycolysis

  • 3) transfer of a hydride ion to NAD+, forming NADH

The Chemical Logic of Glycolytic Pathway

Glycolysis: Step 1

• D-glucose (starting molecule)

• Hexokinase (enzyme)

• Glucose 6-phosphate (product)

• Phosphorylation (reaction type)

  • Phosphate group is transferred from one ATP molecule to the C6-oxygen atom

  • Irreversible reaction

    • First step of a pathway = committed step

  • “Investment” of ATP

• 

  • Regulated step

  • Regulatory enzyme

  • Irreversible reaction

Glycolysis: Step 2

• Glucose 6-phosphate (from step 1)

• Phosphoglucose isomerase (enzyme)

• Fructose 6-phosphate (product)

• Isomerization (reaction type)

  • Glucose 6-phosphate and Fructose 6-phosphate are isomers

• Reversible reaction

• 

  • reversible reaction

Glycolysis Step 3

• Fructose 6-phosphate (from step 2)

• Phosphofructokinase-1 (PFK-1) (enzyme)

• Fructose 1,6-bisphosphate (product)

• Phosphorylation (reaction type)

• Irreversible reaction

• Step sets up “splitting the molecule” from one C6 piece to two C3 pieces

  • “Investment” of ATP

• 

  • Regulated step

  • Regulatory enzyme

  • Irreversible reaction

  • AMP, and Fructose

Glycolysis: Enzyme PFK-1

• PFK-1 is a regulated enzyme

• PFK-1 is activated by ADP, AMP, cAMP, or fructose 2,6-bisphosphate (most potent activator)

• PFK-1 is inhibited by ATP and citrate

• When ATP levels are high, glycolysis is inhibited

• when ATP levels are low, glycolysis is activated

  • WHY?

  • Levels of fructose 2,6-bisphosphate (produced elsewhere in the cell) are connected with insulin and glucagon

  • Indirect influence on glycolysis

• 

Glycolysis Step 4

• Fructose 1,6-bisphosphate (from step 3)

• Aldolase (enzyme)

  • Aldol groups are present on the substrate

  • Aldolase can create or break the aldol functional group

  • Aldol cleavage (reaction type)

• Glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP)

  • Each has 3 carbons

• 

Glycolysis Step 5

• Interconversion of G3P (aldehyde) and DHAP (ketone) occurs

  • Isomerization reaction

• Both G3P and DHAP are monosaccharides

  • Triose

• Triose phosphate isomerase (enzyme)

• Two molecules of G3P are produced from one molecule of glucose

  • More DHAP gets converted to G3P

  • Further metabolism of G3P occurs and continues onward

• 

Glycolysis Step 6

• Glyceraldehyde-3-phosphate (G3P)

  • From step 5

• Glyceraldehyde-3-phosphate dehydrogenase (enzyme)

• 1,3-bisphosphoglycerate (1,3-BPG)

  • Product

• Phosphorylation reaction

  • Phosphate loaded

  • No ATP was invested!

• NAD+ interconverts with NADH

  • Only if aerobic glycolysis

• Redox reaction

• 

Glycolysis Step 7

• 1,3-Bisphosphoglycerate (from step 6)

• Phosphoglycerate kinase (enzyme)

• 3-phosphoglycerate (3-PG)

• Phosphorylation reaction

• ADP to form ATP

  • 1 ATP for every G3P formed

• 1,3-BPG has high energy because it has two phosphate groups

• 

  • Note: Most 2,3-BPG is converted to 3-PG, but some escape; they cause hemoglobin to unload O2 to the muscle

Glycolysis Step 8

• 3-phosphoglycerate (3-PG)

  • From step 7

• Phosphoglycerate mutase (enzyme)

• 2-phosphoglycerate (2-PG)

  • Product

• Phosphoryl group shifts

  • Isomerization (reaction type)

• Swaps the phosphate group*

• 

  • reversible reaction

Glycolysis Step 9

• 2-phosphoglycerate (from step 8)

• Enolase (enzyme)

• Phospho-enol pyruvate (PEP)

  • Not phosphenol

  • Product

• H2O is lost in the reaction

• 

• 

Glycolysis Step 10

• PEP (starting material) has a high energy phosphate group

• Pyruvate kinase (enzyme) catalyzes the transfer of PEP’s phosphate group to ADP

• Irreversible reaction

• Final product of glycolysis is pyruvate

• Forms 1 molecule of ATP (for every molecule of G3P entering the energy producing phase)

• 

Glycolysis Overview

Energetics of Glycolysis

• Energy Investment Phase

• 2 ATP molecules are used to initiate glycolysis

• “Payoff” Phase

  • 2 ATP molecules are produced from one molecule of 3-PG

  • 4 ATP molecules are produced from one molecule of glucose

  • 2 NADH molecules are created and enter the Electron Transport Chain

    • Assuming aerobic conditions

  • 1 NADH = 3 ATP; 2 NADH = 6 ATP

• 

Hormonal Regulation of Glycolysis

• In the well-fed state (carb-rich), insulin levels rise and glucagon levels drop

• Reverse occurs in the starved state (carb-poor)

• Regular consumption of carbs initiates an increase in insulin levels

• Increases the conversion of glucose to pyruvate by activating

  • Hexokinase (Step 1)

  • Phosphofructokinase (Step 3)

  • Pyruvate kinase (Step 10)

• 

Lactate Fermentation

• In anerobic respiration, pyruvate is reduced to lactate by lactate dehydrogenase (enzyme)

  • Ketone (Pyruvate) is reduced to an alcohol (lactate)

• Pyruvate is converted to lactate in:

  • Poorly vascularized tissue or cells lacking mitochondria

  • Skeletal muscle during intense exercise; cramps occur as a result of lactate accumulation

• The production of NAD+ assists in continued glycolysis

  • Step 6

  • Cori cycle (we’ll discuss this later)

• 

Alternate fates of Pyruvate

• oxidative decarboxylation of pyruvate to acetyl CoA

  • 

• Carboxylation of pyruvate to oxaloacetate (OAA)

  • 

• Reduction of pyruvate to ethanol

  • 

Gluconeogenesis

Gluconeogenesis

• Certain tissues need a continuous supply of glucose

  • Brain, erythrocytes, kidney, cornea, exercising muscle

  • Hepatic stores of glycogen are used (glycogenolysis)

• Gluconeogenesis: Synthesis of glucose from non-carbohydrate precursors

  • Lactate, pyruvate, triacylglycerols (fatty acid source)

  • Occurs mostly in liver, and to a limited extent in the kidney

  • Does not occur in the muscle, nerve cells

Gluconeogenesis

• Pyruvate is converted to Oxaloacetate (OAA)

• OAA forms 2-phosphoenolpyruvate via malate

• 

  • 1. Pyruvate carboxylase

  • 2. Malate dehydrogenase (reduction of OAA to malate)

  • 3. Malate dehydrogenase (oxidation of malate to oxaloacetate)

  • 4. PEP carboxykinase (decarboxylation, then phosphorylation)

• Reaction occurs in mitochondria (involves Electron Transport Chain)

  • Pyruvate to Phosphoenol pyruvate via Oxaloacetate (OAA; step 1) and Malate (step 2)

  • 

  • 

Gluconeogenesis: Steps 5 to 8

• 2-Phosphoenol pyruvate is converted to fructose 1,6-bisphosphate

• Glyceraldehyde 3-phosphate: 3 carbon intermediate

  • Isomerizes to form DHAP

• “Reverse reactions” of glycolysis

• 

Gluconeogenesis: Steps 9 to 10

• 2-Phospho-enol pyruvate is converted to fructose 1,6-bisphosphate

• G3P and DHAP condense to form fructose 1,6-bisphosphate

• Fructose 1,6-bisphosphate is converted to glucose

• 

Gluconeogenesis: Steps 11 to 13

• Step 11

  • Fructose-1,6-bisphosphate (from step 10)

  • Fructose-6-phosphate (product)

  • Fructose 1,6-bisphosphatase (enzyme)

  • Dephosphorylation (reaction type)

  • The analogous “reverse” reaction in glycolysis was catalyzed by PFK-1

    • This is bypassed in gluconeogenesis

  • Fructose-2,6-bisphosphate activates PFK-1 (glycolysis)

  • Fructose-2,6-bisphosphate inhibits fructose 1,6- bisphosphatase (gluconeogenesis)

  • 

• Steps 12 and 13

  • Step 12: Fructose 6-phosphate is isomerized to Glucose 6-phosphate

    • Phosphoglucose isomerase (enzyme)

  • Step 13: Glucose 6-phosphate is dephosphorylated to D-glucose

    • Glucose 6-phosphatase (enzyme)

    • Hexokinase (Step 1 of glycolysis) is not involved

    • Final step of gluconeogenesis

  • The release of free glucose from glucose 6-phosphate occurs only in the liver and kidney

    • Gluconeogenesis does not occur in the muscle

    • Muscle lacks glucose 6-phosphatase

Energetics of Gluconeogenesis

• Gluconeogenesis is an anabolic process

• 4 ATP, 2 GTP, 2 NADH are used in gluconeogenesis

  • ATP and GTP are energetically equivalent

  • Energy resides in the phospho-anhydride bond

• 4 ATP, 2 NADH are produced in glycolysis

  • In the payoff phase

• The difference in extra energy is needed to drive gluconeogenesis

Regulation of Glucogenesis

• Pyruvate is the feedstock substrate for other cellular energy pathways

  • Pyruvate carboxylase (enzyme) helps convert pyruvate to oxaloacetate (OAA)*, which is a part of the TCA (Krebs) cycle

  • Pyruvate dehydrogenase (enzyme) catalyzes conversion of pyruvate to acetyl CoA*, the preparatory step in the TCA cycle

• Therefore, pyruvate conversion is regulated

• *Alternate fates of pyruvate

• 

• Fructose 1,6-bisphosphate is dephosphorylated to fructose 6-phosphate

• Fructose 1,6-bisphosphatase (FBPase-1) (enzyme)

• The reverse reaction in glycolysis is catalyzed by PFK-1

• PFK-1: activated by AMP

• FBPase-1: inhibited by AMP

  • Both enzymes are located in the cytosol

  • Both control opposing pathways

  • Reciprocal regulation

Cori Cycle (aka Lactic Acid Cycle)

• Cori Cycle: The combination of glucose transport to actively working tissues, and the reverse transport of lactate from those tissues back to the liver

• Useful to sustain glucose levels when glucose is unavailable

  • e.g. overnight fasting, starvation

  • 

Steps of the Cori Cycle

• Glucose in the muscle is converted to lactate (anerobic glycolysis)

• Lactate diffuses to the blood and reaches the liver

• Lactate is converted to glucose in the liver (gluconeogenesis)

• Glucose re-enters muscle

• Glycolysis starts