Pyruvate Dehydrogenase Complex Part I

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

Fates of Pyruvate

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
  1. 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

  1. 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

  1. 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

Pyruvate Kinase

  • 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

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

Hexokinase

  • 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

Glycolysis

  • 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 Metabolism Overview Part II

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 Metabolism Overview Part I

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

Glucose Metabolism: Energy Yield

GLYCOLYSIS

  • Occurs in the cytosol
  • Anaerobic
  • Inputs: 1 Glucose
  • Outputs: 2 Pyruvates, 2 ATP, 2 NADH

PYRUVATE DECARBOXYLATION

  • Occurs in the matrix
  • Aerobic
  • Input: 2 Pyruvates
  • Outputs: 2 Acetyl CoA, 2 CO2, 2NADH

CITRIC ACID CYCLE

Kreb’s Cycle of Tricarboxylic Acid Cycle

  • Occurs in the mitochondrial matrix
  • Aerobic
  • Input: 2 Acetyl CoA
  • Output: 4CO2, 2ATP, 6NADH, 2FADH2

OXIDATIVE PHOSPHORYLATION

  • Electron transport
  • Chemiosmosis
  • Aerobic
  • Inputs: 10 NADH, 2FADH2
  • Outputs: 30-34 ATP

Total ATP Output: 30-38 ATP

  • Intertextual variation exists: exact calculations beyond the scope of this tutorial.
  • Approximates yield for both eukaryotes & prokaryotes.

Esophagus & Stomach Histology

ESOPHAGUS

Long, muscular tube delivers food from the pharynx to the stomach.

  • Mucosa layer
    • Comprises stratified squamous epithelium; layers of flattened cells provide protection against physical and chemical damage from the foods, liquids, and salivary juices traveling to the stomach.
    • Muscularis mucosae is also visible in our sample.
  • Submucosal layer
  • Submucosal glands
  • Glands of the submucosal and mucosal layers produce protective and lubricating mucous, which contributes to the protective barrier.
  • Submucosa and mucosa form longitudinal folds in the relaxed esophagus; these folds expand to accommodate foods and liquids during swallowing.
  • Muscularis externa layer
    The composition of the muscularis externa layer changes along its length, as follows:
    • The upper 1/3rd comprises skeletal muscle fibers in both the circular and longitudinal layers;
    • The middle 1/3rd comprises a circular layer of skeletal muscle and a longitudinal layer of smooth muscle;
    • Both layers of the lower 1/3rd, which opens to the stomach, contains smooth muscle fibers.
  • The outermost layer of the esophagus is adventitia; once the esophagus passes through the diaphragm, and is no longer anchored to the body wall, it becomes serosa.

STOMACH

Connects with the esophagus, superiorly and the duodenum, inferiorly.
Regions of the stomach:

  • Cardiac, where the esophagus and stomach meet
  • Fundus, in the upper left corner
  • Body, which is the largest region of the stomach
  • Pyloris, which opens to the duodenum
    Gastric Folds, aka, rugae
  • Line the empty stomach; expand to accommodate foods and liquids during gastric filling
    Histological Details:
  • Surface mucous epithelium
    • These cells secrete mucous and form a physical and chemical barrier to protect the underlying stomach wall from stomach contents. In addition to ingested foods and liquids, the stomach wall is potentially vulnerable to the gastric juices it releases as part of digestion.
  • Pits
    • Invaginations of the surface mucosal epithelium form pits, which open to glands deeper within the mucosa; depending on their location in the stomach, the glands produce mucous and/or gastric juices.
    • Lamina propria is visible in our sample between the pits and glands.
    • Slips of muscularis mucosae can be seen extending into the gastric fold.
      Glands:
  • Cardiac and pyloric glands are primarily mucous-secreting; in other words, they do not produce significant quantities of gastric juices.
    • Cardiac glands are highly coiled at their ends, or bases.
    • Pyloric glands are highly branched.
  • Gastric glands of the fundus and body produce both mucous and gastric juices; they are responsible for chemical digestion.
    • Surface epithelial cells line the pit, and secrete surface mucous.
    • Mucous neck cells also secrete mucous.
    • Parietal cells, which appear as round, bulging, and light pink in the histological sample, are found within the neck and base of the gastric gland. These cells, which are also referred to as oxynitic cells, secrete hydrochloric acid and intrinsic factor (required for vitamin B12 absorption).
    • Stem cells are also found within the neck; these cells play important roles in the constant renewal of gastric mucosal cells.
    • Chief cells reside in the base of the gland, and stain darker due to secretory granules containing pepsinogen, which is a precursor to pepsin for protein digestion.
    • Enteroendocrine cells, which is an umbrella term for a variety of cells that secrete peptide hormones. For example, G cells secrete gastrin, and D cells secrete somatostatin.
    • The cells of the gastric gland lie on a basement membrane, which separates them from the surrounding glands and lamina propria.