GLUCONEOGENESIS

• Synthesis of glucose from non-carbohydrate precursors
• Occurs mostly in the liver and minor process in kidney
• Kidney produces 10% total glucose during overnight fast

ENZYMES UNIQUE TO GLUCONEOGENESIS

1. Pyruvate carboxylase (mitochondrial matrix)

• Converts pyruvate to oxaloacetate
• Requires 1 ATP, biotin and 1 CO2 (ABC Reaction)

2. Phosphoenol carboxykinase (cytosolic and mitochondrial isozymes)

• Converts oxaloacetate to phosphoenolpyruvate (PEP)
• Consumes 1 GTP and releases 1 CO2

Cytosolic PEPCK

• Used in the malate shuttle: shuttles oxaloacetate from mitochondrion to cytosol via malate
• Pathway dominates when pyruvate is the gluconeogenic substrate
• Mitochondrion: oxaloacetate –> malate (consumes 1 NADH)
• Cytosol: malate –> oxaloacetate (releases 1 NADH)
• Released NADH used in G3P synthesis
• Pyruvate substrate uses cytosolic PEPCK: consumes 2 NADH

Mitochondrial PEPCK

• Pathway dominates when lactate is substrate
• Cytosol: lactate –> pyruvate (releases 1 NADH)
• Released NADH used in G3P synthesis
• Mitochondrial PEPCK: oxaloacetate –> PEP (can cross mitochondrial membranes)
• Lactate substrate uses mitochondrial PEPCK: does NOT consume NADH

PEP converted to glyceraldehyde 3-phosphate in 4 reversible reactions: 1 ATP and 1 NADH consumed (double energy inputs, substrates and products)

Glyceraldehyde 3-phosphate reversibly combines w/ DHAP to form fructose 1,6-bisphosphate

3. Fructose 1,6-bisphosphatase (cytosol)

• Produces fructose 6-phosphate
• Consumes 1 H20 and releases 1 Pi

Fructose 6-phosphate reversibly converts to glucose 6-phosphate

4. Glucose 6-phosphatase (ER membrane-bound)

• Enzyme complex with 4 proteins
  i. Transport protein: G6P from cytosol to ER lumen
  ii. Phosphatase: removes Pi from G6P to form glucose (consumes 1 H2O)
  iii. Transport protein: transports glucose to cytosol
  iv. Transport protein: transports Pi to cytosol

FINAL BOOKKEEPING:
2 Pyruvate + 4ATP + 2GTP + 2NADH + 6H20 –> 1 Glucose + 4ADP + 2GDP + 2NAD+ + 6Pi + 6H+
2 Lactate + 4ATP + 2GTP + 6H2O –> 1 Glucose + 4ADP + 2GDP + 6Pi + 6H+

Lactate substrate lacks net NADH requirement

CLINICAL CORRELATION

Avidin

• Protein in egg whites
• Binds biotin very tightly (biotin required by pyruvate carboxylase)
• Consuming large amounts of raw eggs over an extended period of time can produce biotin deficiency
• Symptoms: CNS problems (lethargy)

MECHANISMS OF REGULATION

• Allosteric regulation
• Hormonal regulation
• Substrate availability

ALLOSTERIC REGULATION

Pyruvate carboxylase

• 2Pyruvate + 2CO2 + 2ATP –> 2Oxaloacetate + 2ADP
• Activated by Acetyl CoA (product of FA breakdown, marker of energy abundance & low blood glucose)

Phosphoenolpyruvate carboxykinase (PEPCK)

• 2Oxaloacetate + 2GTP –> 2Phosphoenolpyruvate (PEP) + 2GDP + 2CO2

Corresponding glycolytic reaction

• Pyruvate kinase: 2PEP + 2ADP –> 2Pyruvate + 2ATP
• Inhibited by Acetyl CoA

Fructose 1,6-bisphosphatase-1 (FBP-1)

• Fructose 1,6-BP + H2O –> Fructose 6-P + Pi
• Activated by Citrate (CAC intermediate & marker of energy abundance)
• Inhibited by AMP (marker of low energy) & fructose 2,6-BP (hormonally regulated)

Corresponding glycolytic reaction

• PFK-1: Fructose 6-P + ATP –> Fructose 1,6-BP + ADP
• Inhibited by Citrate
• Activated by AMP & fructose 2,6-BP

SUBSTRATE AVAILABILITY

Glucose 6-phosphatase

• Glucose-6-phosphate + H2O –> Glucose + Pi
• Not allosterically regulated because Km >>> [glucose 6-phosphate]
• Substrate level control

Corresponding glycolytic reaction

• Glucokinase: Glucose + ATP –> Glucose 6-phosphate + ADP

HORMONAL REGULATION

• FBP-2 & PFK-2 are hormonally regulated (PFK-2 inactive when phosphorylated)
• High blood glucose = increased Insulin: glucagon ratio = PFK-2 active
  = increased fructose 2,6-BP = promote glycolysis and inhibits gluconeogenesis
• Low blood glucose = decreased insulin: glucagon ratio = PFK-2 phosphorylated & inactive
  = decreased fructose 2,6-BP = slows glycolysis and removes inhibition from gluconeogenesis
• INSULIN: promotes glycolysis
• GLUCAGON: promotes gluconeogenesis

• Synthesis of glucose from non-carbohydrate precursors
• Occurs mostly in the liver and minor process in kidney
• Kidney produces 10% total glucose during overnight fast

THREE KEY SUBSTRATES

1. Lactate: enters pathway via pyruvate

• Produced by exercising muscle and red blood cells
• Reconverted to pyruvate in liver

2. Glycerol: enters via DHAP

• TAG hydrolyzes to glycerol in adipose tissue
• Liver converts glycerol to DHAP in 2 step reaction

3. Amino acids: enter via pyruvate or citric acid cycle intermediates

• Major source of glucose during extended fast
• Muscle tissue hydrolysis releases glucogenic AA during fast
• AA produce alpha-ketoacids in the liver: enter CAC or gluconeogenesis

ESSENTIAL FUNCTIONS OF GLUCONEOGENESIS

• Clears blood lactate from red blood cells and exercising muscle
• Maintains blood glucose during high fat diet or fast

ENZYMES UNIQUE TO GLUCONEOGENESIS

1. Pyruvate carboxylase (mitochondrial matrix)

• converts pyruvate to oxaloacetate
• Requires 1 ATP, biotin and 1 CO2

2. Phosphoenol carboxykinase (cytosol)

• Preceded by malate shuttle (1 NADH consumed and 1 NADH produced)
• Converts oxaloacetate to phosphoenolpyruvate (PEP)
• Consumes 1 GTP and releases 1 CO2

PEP converted to glyceraldehyde 3-phosphate in 4 reversible reactions: 1 ATP and 1 NADH consumed (double energy inputs, substrates and products)

Glyceraldehyde 3-phosphate reversibly combines w/ DHAP to form fructose 1,6-bisphosphate

3. Fructose 1,6-bisphosphatase (cytosol)

• Produces fructose 6-phosphate
• Consumes 1 H20 and releases 1 Pi

Fructose 6-phosphate reversibly converts to glucose 6-phosphate

4. Glucose 6-phosphatase (ER membrane-bound)

• Translocates glucose 6-phosphate to the ER lumen and removes Pi
• Consumes 1 H2O

ENERGY REQUIREMENTS:
(1 ATP + 1 GTP + 1 NADH + 1 ATP) x 2 = 4 ATP + 2 GTP + 2NADH

INNER MITOCHONDRIAL MEMBRANE

• Contains the ETC, ATP synthase, ADP-ATP transporter, phosphate translocase and more
• Impermeable to small molecules (H+, ATP, ADP & Pi)
• ETC pumps protons across impermeable inner membrane: generates chemiosmotic gradient (proton-motive force)

ATP SYNTHASE STRUCTURE

F0 (c, gamma, and epsilon subunits)

• Cylindrical structure embedded in membrane
• Channel through which H+ flows down gradient

F1 (alpha and beta subunits)

• Sits on top of F0 on matrix side

Stator (a, b, and delta subunits)

• Prevents F1 from rotating as F0 does

ATP SYNTHESIS

1. H+ from intermembrane space enters F0
2. H+ protonates asparagine residue within channel
3. Induces rotation of c-ring

• Electrochemical energy (H+ gradient) converted to mechanical energy (rotation)
• On the matrix side, ADP & Pi bind F1 beta subunit

4. Beta subunit interacts with rotating F0: activates and catalyzes formation of ATP

• ATP released into matrix along with H+ that passes through channel

ADP-ATP TRANSPORTER

• Antiporter
• Driven by the electrical potential across membrane
• Intermembrane space more positive than matrix
• ATP has -4 charge while ADP has -3 charge
• Charge difference favors movement of ATP OUT of negatively charged matrix

PHOSPHATE TRANSLOCASE

• Symporter: pumps H+ & Pi from intermembrane space into matrix
• Driven by pH gradient across inner membrane
• pH greater in matrix (more basic, less H+) and lower in intermembrane space (more acidic, more H)
• Protons & Pi move from intermembrane space into matrix
• For every 4 H+ pumped into matrix: 3 drive ATP synthase & 1 drives Pi transport

CLINICAL CORRELATIONS

Uncouplers

• Proteins that make inner mitochondrial membrane permeable to H+
• Example: 2,4 dinitrophenol (DNP)

Brown adipose tissue (BAT)

• Specialized adipose tissue that facilitates non-shivering thermogenesis
• Contains many mitochondria & uncouplers

Complex I: NADH dehydrogenase

• aka NADH-CoQ reductase
• NADH delivers 2 electrons to the complex I and is oxidized to NAD+

CoQ (aka Q10 and ubiquinone)

• Lipid-soluble
• Mobile carrier
• NOT a protein

Complex II: succinate dehydrogenase

• aka Succinate-CoQ reductase
• Also part of citric acid cycle
• FADH2 delivers two electrons to complex II

Complex III: Cytochrome bc1 complex

• aka CoQ-cytochrome c reductase

Cytochrome C

• Water-soluble
• Mobile carrier

Complex IV: cytochrome c oxidase

• Produces one H2O from 2 H+ plus ½ O2 + 2e-
• Complexes I, III & IV pump H+ from matrix to intermembrane space
  (NOT complex II, cyt. C or CoQ)

COFACTORS

• Complex I – flavin mononucleotide (FMN).
• Complex II – FAD & FeS
• Complex III – Heme (Cyt. B & Cyt. C1) & FeS
• Complex IV – heme (Cyt. A & Cyt. A3) & Cu

REDOX REACTIONS

• NADH (2e-) reduces complex I
• FADH (2e-) reduces complex II
• CoQ (mobile carrier) transports 2e- from complex I & II to complex III
• Complex III (simplified diagram): Cyt. B & Cyt. C1 are e- transport proteins
• Cyt. C1 donates 2e- (1e- at a time) to Cyt. C (mobile-carrier)
• Cyt. C transports 4e- to complex IV (2 molecules of Cyt. C deliver 2e- each)
• Complex IV (simplified diagram w/o Cu centers): Cyt. A (4e-) to Cyt. A3 (4e-) to oxygen
• Oxygen (final e- acceptor) 4e- + 4H+ (from matrix) + O2 –> H2O

3 BYPASS REACTIONS

• Bypass complex I & produce less ATP

1. Succinate delivers e- to complex II via FADH2
2. Acyl CoA dehydrogenase (on matrix side) oxidizes fatty acyl CoA & produces FADH2
3. Cytosolic NADH delivers e- via glycerol-3-phosphate

• Enzyme glycerol-3-phosphate dehydrogenase (on intermembrane side) produces FADH2

CHEMIOSMOTIC GRADIENT

• Electrical gradient: from less positive in the matrix to more positive in the intermembrane space.
• pH gradient: from a lower pH in the intermembrane space to a higher pH in the matrix.

Electron transport chain

• Series of controlled redox reactions; pumps H+ into inter-membrane space

Chemiosmosis

• Couples e- transport w/ ATP synthesis

ELECTRON TRANSPORT CHAIN

Complex I: NADH dehydrogenase

• aka NADH-CoQ reductase
• NADH delivers 2 electrons to the complex I and is oxidized to NAD+

CoQ (aka Q10 and ubiquinone)

• Lipid-soluble
• Mobile carrier
• NOT a protein

Complex II: succinate dehydrogenase

• aka Succinate-CoQ reductase
• Also part of citric acid cycle
• FADH2 delivers two electrons to complex II

Complex III: Cytochrome bc1 complex

• aka CoQ-cytochrome c reductase

Cytochrome C

• Water-soluble
• Mobile carrier

Complex IV: cytochrome c oxidase

• Produces one H2O from 2 H+ plus ½ O2 + 2e-
• Complexes I, III & IV pump H+ from matrix to inter-membrane space
  (NOT complex II, cyt. C or CoQ)

CHEMIOSMOSIS

Complex V: ATP synthase

• Inner mitochondrial membrane IMPERMEABLE to most small molecules
• H+ that is pumped across membrane cannot diffuse back through the bilayer
• H+ diffuses down gradient through ATP synthase into the matrix
• Produces 30-34 ATP per glucose molecule (NADH = 3 ATP, FADH2 = 2 ATP

INTERMEDIATES OF THE CITRIC ACID CYCLE

• Oxaloacetate + acetyl-CoA
• Citrate
• Isocitrate
• α-ketoglutarate
• Succinyl CoA
• Succinate
• Fumarate
• Malate

Oh, Can I Keep Studying Science For Med-school

ENZYMES OF THE CITRIC ACID CYCLE

• Citrate synthase (irreversible)
• Aconitase
• Isocitrate dehydrogenase (irreversible) (NADH produced, CO2 released)
• α-ketoglutarate dehydrogenase ( irreversible) (NADH produced, CO2 released)
• Succinyl CoA thiokinase (GTP produced, CoA released)
• Succinate dehydrogenase (FADH2 produced)
• Fumarase
• Malate dehydrogenase (NADH produced)

FINAL BOOKKEEPING

Acetyl-CoA +3NAD+ + FAD + GDP + Pi + 2H20 –> 2 CO2 + 3NADH + 3H+ + FADH2 + ATP + CoA

ITRIC ACID CYCLE (aka Krebs cycle and tricarboxylic acid cycle)

• Occurs under aerobic conditions (has many exits and entry points)
• Occurs in the mitochondrial matrix (pyruvate transported from cytosol)
• First intermediate is Acetyl CoA (two carbon molecule)
• 8 more intermediates to complete the cycle: Oh, Can I Keep Studying Science For Med-school?

ACETYL CoA

• 2-carbon molecule
• Can come from carbohydrates (via pyruvate), fatty acids and amino acids
• Pyruvate dehydrogenase complex: Pyruvate + NAD + –> Acetyl CoA + CO2 + NADH

CITRATE AND ISOCITRATE

• 6-carbon molecules
• Oxaloacetate (4 carbons) + Acetyl CoA (2 carbons) = Citrate
• Isomerized to form isocitrate

ALPHA-KETOGLUTARATE

• 5-carbon molecule
• 1 carbon dioxide and 1NADH released in its production

SUCCINYL CoA

• 4-carbon molecule
• 1 carbon dioxide and 1 NADH released in its production

SUCCINATE

• 4-carbon molecule
• Lost CoA and 1 ATP produced via substrate level phosphorylation

FUMARATE

• 4-carbon molecule
• FADH2 released in its production (F for FADH2 and Fumarate)

MALATE TO OXALOACETATE

• Last reaction in the cycle
• Both are 4-carbon molecules
• Last NADH released in the cycle

TOTAL OUTPUT:

• Pyruvate decarboxylation: 1 NADH and 1 carbon dioxide
  (x2 per glucose)
• Per turn: 2 carbon dioxide molecules, 3 NADH and 2 FADH2
  (2 turns per glucose)
  *NADH and FADH2 deliver electrons to electron transport chain on inner mitochondrial membrane

PYRUVATE DEHYDROGENASE COMPLEX (PDC)

• Pyruvate + CoA + NAD+ –> Acetyl CoA + CO2 + NADH
• Located in mitochondrial matrix
• Irreversible reaction

PDC ENZYMES

E1, pyruvate dehydrogenase/pyruvate decarboxylase

• Catalyzes pyruvate to acetyl (releases CO2)
• Cofactor: thiamine pyrophosphate (Vitamin B1)

E2, dihydrolipoyl transacetylase

• Attaches CoA to acetyl
• Cofactor: lipoic acid (not vitamin-derived) & coenzyme A (pantothenic acid/vitamin B5)

E3, dihydrolipoyl dehydrogenase

• Reduces NAD+ to NADH
• Cofactor: NAD+ (niacin/vitamin B3) & FAD (riboflavin/vitamin B2)

Lipoic acid is only cofactor for PDC that is not vitamin-derived

CLINICAL CORRELATION

PDC-based pathology

• Deficiencies in vitamins or PDC cofactors produce initial neurological/muscular symptoms

KEY FATES OF PYRUVATE

1. Acetyl CoA: substrate for citric acid cycle and fatty acid synthesis
2. Oxaloacetate: intermediate in CAC and substrate for gluconeogenesis
3. Lactate: produced by eukaryotes in absence of oxygen
4. Ethanol: produced by yeast and some bacteria (including intestinal flora) in absence of oxygen.

AEROBIC CONDITIONS

1. Cellular respiration: Pyruvate converts to acetyl CoA

• Fed conditions (glucose abundant)
• Occurs in mitochondrial matrix
• Pyruvate dehydrogenase complex
• Irreversible reaction: produces 1 CO2 and 1NADH
• Acetyl CoA enters the citric acid cycle and oxidative phosphorylation
• Final product is ATP

2. Gluconeogenesis: Pyruvate converts to oxaloacetate

• Fasting conditions (glucose in demand)
• Occurs in liver (minor process in kidneys): mitochondrial matrix
• Pyruvate carboxylase
• Irreversible reaction
• Oxaloacetate is substrate for gluconeogenesis and CAC intermediate

ANAEROBIC CONDITIONS

3. Lactic acid fermentation (humans)

• Occurs in exercising muscle and red blood cells: cytosol
• Glycolysis: 1 glucose = 2 pyruvates + 2 ATP + 2 NADH
• Lactate dehydrogenase: 2 pyruvate + 2NADH = 2 lactate + 2 NAD+
• Reversible reaction
• Lactate can enter bloodstream and travel to liver: lactate dehydrogenase catalyzes reverse reaction (lactate to pyruvate)

Clinical correlation: intense exercise can produce lactic acidosis; lactate accumulates in muscle cells and causes intracellular drop in pH

4. Ethanol production (yeast and select bacteria)

• Can occur in inteestinal flora
• Glycolysis: 1 glucose = 2 pyruvates + 2 ATP + 2 NADH
• 2 step rxn: pyruvate to acetaldehyde to ethanol
• Ethanol formation consumes 2 NADH in second step and produces 2 NAD+ for reuse
• Irreversible reaction
• Fermentation in yeast used to make beer and wine