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

• Last enzyme in glycolysis
• Irreversibly dephosphorylates phosphoenolpyruvate (PEP) to form pyruvate
• 1 ATP produced by substrate level phosphorylation
• Several isozymes: M-type (muscle) and L-type (liver)
• All isozymes allosterically regulated (L-type also hormonally regulated)

M-TYPE ISOZYMES

Allosteric regulation

Activation

• AMP: marker of ATP depletion or low energy
• Fructose 1,6-bisphosphate: product of rate-limiting reaction in glycolysis
  (feed-forward activation: stimulates downstream glycolytic enzymes)

Inhibition

• ATP: sufficient energy
• Acetyl CoA: first intermediate of citric acid cycle
• Alanine: can be produced from pyruvate; sufficient pyruvate in the cell

L-TYPE ISOZYME

• Allosteric and hormonal regulation (similar to PFK-2)

Hormonal regulation

Activation

• Insulin activates phosphatases, which remove phosphate from PK
• Makes PK susceptible to positive allosteric regulators

Inhibition

• Glucagon promotes phosphorylation of PK via cAMP-dependent pathway
• Makes PK susceptible to negative allosteric regulators

CLINICAL CORRELATION

Pyruvate kinase deficiency

• Produce hemolytic anemia (spiculated RBC’s)
• RBC biconcave shape maintained by sodium-potassium pumps (require ATP)
• RBC’s do not have mitochondria: rely on glycolysis for ATP

PHOSPHOFRUCTOKINASE-1 (PFK-1)

• Catalyzes rate-limiting step in glycolysis
• Catalyzes irreversible phosphorylation of F6P to F1,6P.
• Allosterically regulated (hormonally regulated in liver)

PFK-1 INHIBITION

• Citrate, intermediate of citric acid cycle
• ATP, final product of glycolysis and cellular respiration
• H+, symptom of lactic acid buildup in exercising muscle

PFK-1 ACTIVATION

• AMP, marker of ATP depletion.
• Fructose-2,6-bisphosphate (special case)

PFK-2/FBP-2 (BIFUNCTIONAL ENZYME)

• PFK-2: F6P –> F2,6P (activates PFK-1)
• FBP-2 (fructose-2,6-bisphosphatase): F2,6P –> F6P (deactivates PFK-1)
• PFK-2/FBP-2 regulated differently in different tissues

Skeletal muscle

• Feed-forward activation
• Substrate-level regulation: F6P
• When F6P HIGH: FBP-2 inactive and PFK-2 active (activate PFK-1)
• When F6P LOW: FBP-2 active and PFK-2 inactive (inhibits PFK-1 activity)

Liver

• Hormonal regulation: PFK-2 has phosphorylation site (unlike muscle)
• PFK-2 is INACTIVE when phosphorylated
• Glucagon activates protein kinase A (PKA), which phosphorylates PFK-2
• Insulin activates phosphoprotein phosphatase (PPP), which dephosphorylates PFK-2

FED STATE

• HIGH blood glucose
• INSULIN secreted –> PPP activated –> PFK-2 dephosphorylated (ACTIVE)
• HIGH F2,6P activates PFK-1
• Promotes glycolysis

FAST

• LOW blood glucose
• GLUCAGON secreted –> PKA activated –> PFK-2 phosphorylated (INACTIVE)
• LOW F2,6P deactivates PFK-1
• NO glycolysis

Liver responds to entire body’s glucose needs

• Site of gluconeogenesis: glucose synthesized from non-carbohydrate precursors and released into the bloodstream

• Catalyzes phosphorylation of glucose to form glucose-6P
• Traps glucose inside the cell
• 1st regulated enzymes in glycolysis – catalyze an irreversible reaction
• 4 isozymes – I, II, III, and IV (glucokinase)

HEXOKINASE (I, II & III) vs. GLUCOKINASE (IV)

• Tissue distribution
• Kinetics (Km and Vmax)
• Regulation (allosteric vs hormonal)

Hexokinase

• Ubiquitous in mammals
• Low Km & low Vmax
• Allosteric regulation – inhibited by glucose-6P

Glucokinase

• Liver & pancreatic beta cells
• High Km & high Vmax: spares glucose for brain, muscle & other tissues (glucose sensor)
• Hormonal regulation: inhibited by glucagon, activated by insulin
• Glucokinase regulatory protein (GKRP): nuclear protein that reversibly binds/inactivates glucokinase
• High [glucose] inhibits GKRP & promotes glucokinase release
• Fructose-6P (glycolytic intermediate in equilibrium with glucose-6P): promotes GKRP-GK binding
• Liver glucose-6P shunts into one of three pathways: glycolysis, glycogen or fatty acid synthesis

• 1 glucose (6-carbon sugar) breaks down to 2 pyruvates (3-carbon sugar).
• Net 2 ATP produced: 2 consumed (investment phase) and 4 generated (pay off)
• 2 NADH produced

REGULATED STEPS

Hexokinase

• Glucose + ATP Glucose-6-phosphate + ADP

Phosphofructokinase

• Fructose-6-phosphate + ATP → fructose-1,6-biphosphate + ADP

Pyruvate kinase

• Phosphoenol pyruvate + ADP → Pyruvate + ATP

ENZYMES OF GLYCOLYSIS
Listed in chronological order (substrate/product in parentheses)

• Hexokinase (glucose/glucose-6P)
• Phosphoglucose isomerase (glucose-6P/fructose-6P)
• Phosphofructokinase (fructose-6P/fructose-1,6P)
• Aldolase (fructose-1,6P/G3P & DHAP)
• Triose phosphate isomerase (DHAP/G3P)
• Glyceraldehyde-3-phosphate dehydrogenase (G3P/1,3-bisphosphoglycerate)
• Phosphoglycerate kinase (1,3-bisphosphoglycerate /3-phosphoglycerate)
• Phosphoglycerate mutase (3-phosphoglycerate /2-phosphoglycerate)
• Enolase (2-phosphoglycerate /phosphoenol pyruvate)
• Pyruvate kinase (phosphoenol pyruvate /pyruvate)

GLUCOSE OXIDATION EQUATION
Glucose + 6 O2 –> 6 CO2 + 6 H2O + Energy (ATP + heat)

• Most energy is generated in mitochondrial matrix

Common Abbreviations:

• ATP: adenosine triphosphate
• NADH: nicotinamide adenine dinucleotide
• FADH2: flavin adenine dinucleotide
• CoA: Coenzyme A

KEY PROCESSES IN GLUCOSE OXIDATION

• Glycolysis
• Pyruvate decarboxylation
• Citric acid cycle (also known as the Krebs’ cycle and the tri-carboxylic acid (TCA) cycle)
• Oxidative phosphorylation (electron transport chain & chemiosmosis)

CITRIC ACID CYCLE

• 1 glucose molecule requires 2 citric acid cycle turns
• Input for each turn: 1 Acetyl CoA
• Output for each turn: 3 NADH + 2 CO2 + 1 ATP + 1 FADH2
• NADH & FADH2: electron transfer molecules for oxidative phosphorylation
• Occurs in mitochondrial matrix

Substrate level phosphorylation

• ATP generated from substrates in glycolysis and citric acid cycle
• NOT from oxidative phosphorylation via NADH or FADH2

OXIDATIVE PHOSPHORYLATION

• Input: 10 NADH + 2 FADH2 (from glycolysis, pyruvate decarboxylation & CAC)
• Generates 30-34 molecules of ATP per glucose
• Main energy-generating process in respiration
• Comprises electron transport chain and chemiosmosis
• Occurs on inner mitochondrial membrane

Electron transport chain

• Series of redox reactions
• Pumps proton from matrix into intermembrane space
• Generates electrochemical gradient for ATP synthesis via ATP synthase
• Contains several protein complexes (I through IV)
• NADH gives electrons to complex I
• FADH2 gives electrons to complex II
• Complex I, III, and IV pump H+ into intermembrane space (Complex II DOES NOT)
• Complex IV consumes 1 O2 (final e- acceptor) to produce 2 H2O

Chemiosmosis

• ATP synthase: harnesses energy in electrochemical gradient (generated by ETC) to synthesize ATP from ADP & Pi

GLUCOSE OXIDATION EQUATION
Glucose + 6 O2 –> 6 CO2 + 6 H2O + Energy (ATP + heat)

• Most energy is generated in mitochondrial matrix

Common Abbreviations:

• ATP: adenosine triphosphate
• NADH: nicotinamide adenine dinucleotide
• FADH2: flavin adenine dinucleotide
• CoA: Coenzyme A

KEY PROCESSES IN GLUCOSE OXIDATION

• Glycolysis
• Pyruvate decarboxylation
• Citric acid cycle (also known as the Krebs’ cycle and the tri-carboxylic acid (TCA) cycle)
• Oxidative phosphorylation (electron transport chain & chemiosmosis)

GLYCOLYSIS

• 1 glucose –> 2 pyruvate + 2 ATP + 2 NADH
• Anaerobic reaction: no O2 required
• Occurs in cytosol

Substrate level phosphorylation

• ATP generated from substrates in glycolysis and citric acid cycle
• NOT from oxidative phosphorylation via NADH or FADH2

PYRUVATE DECARBOXYLATION

• Pyruvate + CoA + NAD+ –> Acetyl-CoA + CO2 + NADH
• Occurs in mitochondrial matrix