Lecture: 15 Cofactors
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Enzyme Cofactor deficiencies:
o Homocystinuria – vitamin B6/Vitamin B12/Folate (multi-systemic disorder;
accumulation of the amino acid homocysteine in the serum)
o Pellagra – niacin (diarrhea, dermatitis, dementia, death)
o Scurvy – Vitamin C (general weakness, anemia, gum disease, and skin hemorrhages)
o Ricketts – Vitamin D (defective mineralization before epiphyseal closure; leading to
fractures and deformities)
Cofactor Classification
o
o
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Loosely bound – molecule or ion will bind to the enzyme, carry out reaction, then
dissociate form the enzyme
 ATP; Magnesium ions
Tightly bound – may be covalently attached to an enzyme residue in the active site or
bind non-covalently with very high affinity
 Heme, biotin and lipoic acid (covalently)
 Flavins bind zinc ions (non-covalently)
Vitamin-derived coenzymes
 Some vitamins utilized as is when consumed (Vitamin C)
 Most vitamins are transformed before they are used (panthothenic acid making
Coenzyme A)
Nicotinamide Coenzymes
 Used in reactions involving hydride ions (NADH/NADPH)
 Donates or accepts hydride ion; commonly used in dehydrogenase enzymes
 NADH – carry electrons to mitochondria to drive ATP production
 NADH + Pyruvate  lactate and NAD+
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NADPH – used in fatty acid and cholesterol syntheses
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Flavins – carry electrons as reducing agents
 Electrons and protons transferred in two steps
 FMN/FAD – carry electons to mitochondria to drive ATP production
 FMN – flavin mononucleotide
 FAD – flavin adenine dinucleotide
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Coenzyme A – used as an acyl group carrier (acetate, fatty acid or ketone group)
o Sulfur end is the reactive end
o Used in synthesis and oxidation of fatty acids and the oxidation of pyruvate in the citric
acid cycle
o
Thiamine Pyrophosphate – energy metabolism, essential for neuronal and neurocognitive
function (used in synthesis of acetylcholine, nucleic acids, and NADPH)
o Performs decarboxylation and transketolation reactions: transfer of aldehyde groups
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Carbon in ring is a very strong nucleophile it activates substrates to be more
nucleophilic; no side chain in any protein has this power
o
Pyridoxyl Phosphate(PLP) – Vitamin B6 derivative, covalently attached to host enzyme
o Carries out reactions with amino acids like isomerizations, decarboxylations, and side
chain removals
o Function depends on NADH, coabalamin, folic acid
o
Biotin – vitamin used as-is, covalently attached to lysine in an enzyme; used in hair and nails
o Carboxyl transfer reactions and ATP-dependent carboxylations
o Important for: transforming Phosphoenol pyruvate to oxaloacetate(citric acid cyle);
transforming acetyl CoA to malonyl CoA for fatty acid synthesis
o
Tetrahydrofolate – provides transfer of one carbon unit as CH3, CH2, or CH
o Two nitrogens are the site of function
o Critical vitamin for:
o
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Lipoamide – formed from lipoic acid (becomes lipoamide when covalently attached to protein
Lys side chain)
o Acyl group carrier
o Redox properties:
 Ring form with S-S bond is oxidized form
 Ring form with –SH group is reduced form
 NADH is produced from NAD+ when lipoamide is oxidized
o
Cobalamin (Vitamin B12)
o Adenosyclobalamin – catalyzes exchange reactions (isomerizations)
o Methylcobalamin – transfers methyl groups
Lipid-Soluble Vitamins
o Vitamin A – used in vision
o Vitamin K – used in synthesis of blood clotting proteins
o Vitamin E – anti-oxidant
o Vitamin D – Calcium uptake
Heme – key component of hemoglobin and enzymes in electron transport chain
o Prosthetic group tightly or covalently bound to host protein
Tightly-Bound Metals
o Iron-Sulfur clusters – electron transfer reactions, especially in electron transport chain
and in TCA cycle
o Zinc ions – often form electrostatic interactions with substrates to control position of
substrate or to activate a bond or group
Lecture 16: Glycoproteins
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The Glycocalyx – network of glycoproteins and proteoglycans that extends outwards from the
surface of the cell
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Protects the outer surface of the cell from mechanical disruption; allows cell to attach
itself to other surfaces
o Unique to every individual, serves as a mechanism to identify self from foreign
o Cells with glycocalyx:
 Mature platelets – allows them to adhere to injury site
 Vascular endothelial cells – reduces friction of blood flow
 Sperm cells – allows them to adhere to ovum
 Fertilized ovum – allows it to adhere to uterine linig
 Cancer cells – have unique glycocalyx (allows them to be identified against
normal cells)
Glycosaminoglycans (GAGs) – heteroglycans with linear, regular repeating disaccharide units
o Five classes:
 Hyaluronic acid (HA)
 Chondroitin sulfates (CS)
 Heparin and heparin sulfates (HS)
 Keratin sulfate (KS)
 Dermatin sulfate (DS)
o One of the monosaccharide is always an amino sugar: Glucosamine or galactosamine
o Other monosaccharide is alduronic acid or its isomer
o Multiple sulfations allow sequences to have specific recognition functions
o GAGs are attached to core proteins to form proteoglycan
o Link trisaccharides are always the same (Galactose, Galactose, Xylose)
o
Proteoglycan
o Highly variable
o Excreted from cells; key component of extracellular matrix (interacts with proteins of
matrix: collagen, fibronectin, laminin)
o Found in synovial fluid of joints, vitreous humor of the eye, arterial walls, bone and
cartilage
o Forms a meshwork that limits exposure of cell surface to various agents
o Acts as a lubricant and as shock absorber (can expand and compress easily)
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Bottle-Brush structure – presence of many negative charges leads to charge repulsion
and GAGs being very extended molecules
Glycoproteins – may be N-link or O-linked
o N-linked saccharides are attached via the amide nitrogen of Asn side chain
o O-linked saccharides are attached to hydroxyl group of serine, threonine, or
hydroxylysine
o Examples of glycoproteins
 O-linked – simpler (usually 1-3 sugars)
 Carbohydrate affects recognition and structure
 Forces protein to adopt an extended conformation mostly
 Believed to: 1) keep agents away from surface or 2) make it easier for
agents to find a receptor above the complicated extracellular matrix
 N-linked – complex, highly varied structure
 Structure signals location and function
 Alter the chemical and physical properties of proteins
 Stabilize protein conformations and/or protect against proteolysis
 Cleavage of monosaccharide units from N-linked glycoproteins in blood
targets them for degradation in the liver
GAGs and Disease – can be recycled and used for new synthesis
o Defective or deficient enzymes to break down GAG’s causes them to build up inside
cells, organs and blood which causes cellular damage, dysfunctional development and
often retardation in children
The Extracellular Matrix – fibrous proteins embedded in amorphous “ground substance”
o Ground substance
 Hyaluronic acid – huge hydrated GAG polysaccharide
 Proteoglycans
 Adhesive proteins – glues ECM components to cell membrane
 Mainly laminin and fibronectin
o Integrins
 Integral membrane proteins in plasma membrane that binds adhesive proteins
 Span membrane, so provide contact/communication between ECM and
cytoskeleton
o Matrix metalloproteinases (MMPs)
 Enzymes that cleave one or more components of the ECM
 Responsible for normal and pathological remodeling
 Cancerous cells have increased MMP activity that breaks ECM barrier so
that cancer cells can migrate
o Fibrous proteins provide tensile strength and elasticity
o Ground substance provides deformability, resilience, cohesion
The Cytoskeleton
o Protein scaffolding consists of actin filaments and microtubules
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o Maintains cell shape
o Provides framework for transporting cell components around cell
o Maintains complex array of subcellular organelles
o Dynamic structure – can change in response to cellular signals
o Key for cell movement
Disorders related to ECM
o Chronic Obstructive Pulmonary Disorder (COPD) – Fibrosis due to thickening of ECM
o Ehlers-Danlos syndrome – error in collagen fibril assembly
o Thin-basement membrane nephropathy/Alport syndrome – defect in collagen synthesis
needed to make basement membrane in kidney; basement membrane lose extracellular
matrix protein and fine morphological structure
o Marfan syndrome – defect in fibrillin-1, critical component of ECM and elastic fibers
Lecture: 17 Membrane Transport
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Equivalent – 1 mole = 1 equivalent
o Monovalent ions (Na+, K+) – 1 mole = 1 equivalent (1 equivalent = 1 mole)
o Divalent ions (Ca+2, Mg+2) – 1 mole = 2 equivalents (1 equivalent = ½ mole)
o Trivalent ions (Fe+3, N-3) – 1 mole = 3 equivalents (1 equivalent = 1/3 mole)
o 1 equivalent = 1000 milliequivalents
Membrane Flux – number of units moving across a given area per unit time
o Measured in moles/sec/cm2
o Simple diffusion – freely diffusible molecules can rapidly equilibrate across the
membrane (some gasses, small uncharged symmetrical molecules, water)
 Water needs a protein called aquaporins (AQPs) to flow through
o Facilitated diffusion – charged and polar molecules cannot freely equilibrate and require
specific transport mechanisms
o Substances flow from high concentration to low concentration
 Transporting substances against the electrochemical gradient, energy is
required (active transport)
Electrochemical potential – electrical potential will balance chemical difference
o Membrane potential is Vm (membrane voltage)
 As Vm gets less negative = depolarized
 As Vm gets more negative = hyperpolarized
Simple Diffusion – passive transport; molecules will pass freely through the bilayer unassisted
driven by concentration gradient
o Factors affecting simple diffusion:
 Size must be small
 Uncharged
 Nonpolar – relatively hydrophobic
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Concentration always from higher to lower
Temperature – kinetic energy related to temperature changes
Permeability coefficient – (partition coefficient) how readily the solute dissolves
in the lipid phase of the bilayer; (diffusion coefficient) how readily the solute
traverses lipid phase of bilayer
 Greater the diffusion coefficient the more some substance will pass
through the membrane; Greater the partition coefficient the more a
substance can pass through the membrane; Smaller the distance across
the membrane the more likely the substance can pass through the
membrane
 Rate of diffusion is inversely related to the molecular radius
Facilitated Diffusion – require a specific transporter; uses energy of electrochemical gradient to
drive transport
o Two mechanisms for gating:
 Voltage-dependent – membrane voltage changes, then the channel changes
conformation which opens or closes the channel
 Ligand-dependent – small molecule binds to a channel, then it causes a
conformational change in the channel that opens or closes the channel
o Examples:
 Glucose enters RBC by passive diffusion, once inside glucose is phosphorylated,
which keeps [glucose] inside the cell small
 CO2/HCO3 move in and out of the RBC based on the environmental conditions
Active Transport – requires transporters to transport against the electrochemical gradient;
requires energy input to overcome the positive gibbs free energy
o Examples: ion transport to set up a membrane potential; creating a gradient that will
drive another transport (secondary active transport)
o The Sodium pump – use of ATP to transport 3 Na+ out of the cell, and 2 K+ into the cell
net +1 outside the cell; sets up inward Na+ gradient and outward K+ gradient (important
for nerve transmission, action potentials, and epithelial cell functions)
Cotransport and Secondary Active Transport – the transport of one substance along its
electrochemical gradient can be used to transport another substance against its gradient
o Transport can be in the same direction (symport) or opposite direction (antiport)
o Paired with a primary active transport system to build a gradient
o Example: NA+ gradient built up outside the cell by the sodium pump, which provides
the energy to drive the active transport of glucose into the cell
o Glucose Transporters:
 GLUT 1 – RBC, blood-brain, blood-retinal, blood-placental, blood-testis barriers
 Expressed in cell types with barrier functions; a high-affinity glucose
transport system
 GLUT 2 – liver, kidney, pancreatic β-cell, serosal surface of intestinal mucosa cell
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A high-capacity, low-affinity transporter; may be used as the glucose
sensor in pancreas
GLUT 3 - Brain (neurons)
 Major transporter in the CNS; a high-affinity system
GLUT 4 – Adipose tissue, skeletal muscle, heart muscle
 Insulin-sensitive transporter; increase in insulin = increase in GLUT 4
transporters; a high-affinity system
GLUT 5 – intestinal epithelium, spermatozoa
 Fructose transporter
Lecture: 18 and 20 Cellular Signaling
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Hormone – a substance that is produced in one tissue or organ that is released into the blood
and carried to another tissue or organ where it acts to produce a specific response
o Protein activation, production of second messengers, changes in transcriptional
activities, etc
o Examples of hormones – amino acids and/or their derivatives, peptides, glycoproteins,
cholesterol derivatives, fat-derived molecules
Signal Transduction –
o external chemical signals received by receptors on the outside of plasma membrane
o The receptors produce chemical signals on the inside of the cell
o These chemical signals propagate through the cell where they elicit specific responses
o Some chemical signals enter the cell directly, and bind to an intracellular receptor,
which has direct effects on cell processes (i.e. DNA)
Two major classes of receptors
o Extracellular – typically bin peptide/protein hormones (insulin, growth hormone,
parathyroid hormone) and tyrosine-derived catecholamines (dopamine,
norepinephrine, epinephrine)
 Almost always act to stimulate a signal cascade  proteins activated  second
messengers produced  many cellular proteins affected
o Intracellular – typically bind steroid hormones (glucocorticoids like cortisol;
mineralcorticoids like aldosterone; androgens and estrogens; Vitamin D), retinoic acid
derivatives and thyroid hormones
 Almost always act at the DNA level  altering transcription genes (turn on or
off expression of a given protein, etc.)
Basic Steps in Signaling
o Recognition of the hormone signal – hormone binds to specific receptor
o Transduction of the signal across the membrane – receptor conformational change
o Transmission to intracellular components – receptor activates adaptor proteins
o Modulation of the effector – secondary messengers produced, target proteins activated
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o Response of the cell to the signal – cell status changes
o Termination of the signal – degrade secondary messenger, turn off target proteins
Types of Signaling
o Endocrine – source of hormone and target are far part
o Paracrine – source of hormone and target are adjacent
o Autocrine – cell produces and receives its own signals
Receptor Categories
o Five basic types of receptors
 Ligand-gated ion channels
 G-protein coupled receptors
 Catalytic receptors
 Intracellular (steroid) receptors
 Transmembrane proteins that release transcription factors
o Three types of plasma membrane receptors
 Ion channel receptors
 Receptors that are kinases or bind kinases
 Heptihelical (7-membrane spanning helices) receptors
Acetylcholine Receptor – A ligand-gated ion channel (paracrine)
o Acetylcholine (Ach) binding to Ach receptor stimulates a conformation change in the
receptor
o Opens a channel allowing K+ and Na+ ions to flow  changing membrane polarization
o Signal terminated by acetylcholinesterase (degrades Ach)
Glucagon Receptor – A heptihelical, G-protein coupled receptor (endocrine)
o Glucagon – hormone that signals the fasting state; converts energy stores to fuel stops
buildup of energy stores
o G-protein  adenylate cyclase  cAMP  protein kinase A (PKA)  phosphorylates
specific enzyme of mobilization path
o Phosphorylated fuel utilization enzymes turned on, activity increases; phosphorylated
fuel storages enzymes turned off, inactive
o Signal is quenched by degradation of cAMP
o
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Signaling cascades amplify the hormone signals
o One hormone binds to one receptor
o One receptor activates one G-protein
o One α-subunit activates one adenylate cyclase
o One adenylate cyclase produces lots of cAMP
o cAMP activates lots of PKA
o PKA phosphorylates lots of target proteins
G-protein-coupled receptors
o G-protein catalytic subunit is inactive when GDP is bound
o Hormone binding to receptor  conformational change in receptor  stimulates
exchange of GDP for GTP  activates α-subunit to release βγ complex  βγ complex
activated
o Activated α-subunit  binds to adenylate cyclase  activated adenylate cyclase 
produce cAMP  change in adenylate cyclase  hydrolysis of GTP to GDP  causes αsubunit to leave  rebind to βγ complex and receptor  waits for another receptor
event
cAMP activation of PKA
o cAMP binds to regulatory subunits on PKA, releasing 2 catalytic subunits
o activated C subunits phosphorylate target proteins using ATP
cAMP signal termination
o adenylate cyclase produces cAMP from ATP
o cAMP phosphodiesterase breaks the cyclic bond to form AMP (no signaling properties)
o cAMP phosphodiesterase continuously breaks down cAMP, once adenylate cyclase is
turned off, the remaining cAMP will be consumed by cAMP phosphodiesterase
o decreased cAMP causes PKA regulatory subunit to rebind to catalytic subunit
cAMP mediated Gene transcription
o cAMP response element binding protein (CREB) binds cAMP binding protein (CBP)
Different G-proteins:
o Gs (stimulatory) – activated adenylate cyclase, increases cAMP production, also activates
Ca2+ channels
o Gi (inhibitory) – inhibits adenylate cyclase, prevents cAMP production, also activates K+
channels
o Go – inhibits Ca2+ channels (depresses nerve activity)
o Gq – activates phospholipase C  produces IP3  increases cell Ca2+ and DAG 
activates PKC (smooth muscle contraction)
o G12/13 – cytoskeletal remodeling, smooth muscle contraction
Receptor Kinases
o Composed of two subunits, initially separated (heterodimers or homodimers)
o Hormone binding stimulates association of the two subunits (activating the receptor,
phosphorylating the receptor)
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Can be autophosphorylated (tyrosine kinase receptors) or a kinase that binds
(JAK-STAT receptors)
o Phosphorylated sites become docking sites for adaptor proteins  activated adaptor
proteins leave the receptor and bind cellular targets
The Insulin Receptor
o Insulin binding stimulates the receptor, which autophosphorylates
o Phosphorylated receptor binds the insulin receptor substrate (IRS), which gets
phosphorylated
o Phosyphorylaed IRS then activates target proteins: Grb2, Phospholipase C-γ and
phosphatidyl inositol 3-kinase
Intracellular receptors
o Transactivation – ligand induced conformational change of the receptor that results in
its binding to a specific segment of DNA to initiate or block transcription
o Steroid/thryroid hormone receptors
 Hormone enters cell  binds to receptor causing dimerization 
receptor/hormone binds to specific place on DNA called hormone response
element  activates cis linked genes
Clinical correlation
o Cholera toxin product of bacterium vibrio cholera
 Toxin catalyzes the ADP ribosylation of Gαs inhibiting GTPase causing continuous
activatation of adenylate cyclase in epithelial cells
 Elevated levels of cAMP cause an increase in Cl- conductance and water flow 
large fluid loss
o Pertussis Toxin – causative agent of whooping cough
 Pertussis toxin is unable to inhibit cAMP generation (does not promote
generation of cAMP)
Lecture: 19 Vitamins and Minerals
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Vitamins – essential group of micronutrients
o Organic compounds distinct from fats, carbohydrates or protein
o Natural components of foods, usually present in minute amounts
o Not synthesized by the body in amounts to meet our physiologic needs
o Essential for normal physiologic function (maintenance, growth, etc)
o Cause a specific deficiency syndrome by their absence or insufficiency
o Two groups:
 Fat soluble (non-polar solvents) – A, D, E, K
 Water soluble (polar solvents) – Vitmain C, thiamin, riboflavin, niacin, B6, biotin,
panthothenic acid, folate, B12
Fat-Soluble Vitamins – can be stored
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o Absorption depends on micellar dispersion in the intestine (bile salts)
o Fat required for proper absorption
o Absorbed and transported with lipids
o Partition into the lipid portion of the cell (membrane, lipid droplets)
o Excreted in eterohepatic circulation
Vitamin A – dually works as a hormone
o 11-cis-retinol – part of the visual cycle, vision pigment
o Retinoic acid – growth, reproduction and maintenance of epithelial tissue
o Beta-carotene – precursor of vitamin A, antioxidant properties
o Sources: (humans can store upto 2 years in adipose tissue)
 Vitamin A from animal foods
 Beta-carotenes – plant foods
o Toxicity:
 Retinol – large doses causes harm
 Carotenoids – low risk; stored in adipose tissue (orange skin)
o Deficiency:
 Night blindness (xeropthalmia) (#1 vitamin deficiency in the world)
 Bitot’s spots corneal keratinization (buildup of keratin in conjunctiva)
 Poor wound healing
 Dry skin
 Infection susceptibility
Vitamin D – dually works as a hormone
o stimulate osteoclast activity with parathyroid hormone
o Bone demineralization, calcium absorption in kidney and small intestine
o Sources:
 D2 (ergocalciferol) – absorbed by small intestine in milk
 D3 (cholcalciferol) – synthesized in skin with UV light
 25-hydroxy D3 – storage form in liver
 1,25-dihydroxy D3 – active form in kidney
o Toxicity:
 Can be stored in adipose tissue
 Bone demineralization, hypercalcemia, kidney stones
o Deficiency:
 Children – rickets, skeletal deformation, short stature, ‘pigeon breast’
 Adults – ostemalacia, general bone pain
 Common in decreased sun exposure, fat absorption disorders, liver disease,
chronic renal failure
Vitamn E – Eight forms, alpha-tocopherol most biologocially active (source nuts)
o Anti-oxidant, maintain cell membrane integreity
 It protects unsaturated phospholipids of the membrane from oxidative
degradation from free radicals and highly reactive oxygen species
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Deficiency:
 Fragile membranes, poor vision, neuro dysfunction, myopathy
 At risk include: lipid malabsorption diseases (biliary atresia, exocrine pancreatic
insufficiency, lipid transport abnormalities or cystic fibrosis)
 Takes five or more years for symptoms to develop
Vitamin K – diet (green leafy veggies) and intestinal microbe synthesis
o supplement newborns with Vitamin K due to low placental absorption, low synthesis
from gut bacteria
o clotting factors, cofactor for glutamate carboxylase (calcium binding), factors II, VII, IX, X
(coagulation)
o Deficiency: (diet needs to be stable when using Coumadin)
 Poor clotting, bruising, mucus membrane bleeding
 Elderly – hip fractures
 Recent use of broad spectrum antibiotics
Vitamin B1 (Thiamin) – PDHase, transketolase
o Deficiency:
 Dry beriberi (wasting and partial paralysis from damaged peripheral nerves)
 Wet beriberi (combination of heart failure and weakening of the capillary walls
can cause edema)
 Wernicke-Korsakoff (vision changes, ataxia, and impaired memory)
 Wernicke’s encelphalopathy – low RBC transketolase activity
 Amnesia
Vitamin B2 (riboflavin) – FAD, FMN
o Deficiency:
 Angular cheilitis (inflammation of the corner of the lips)
 Corneal vascularization (blood vessel formation in the cornea)
Vitamin B3 (niacin) – NAD, NADH
o Deficiency:
 No appetite, weakness, dermatitis, dementia, diarrhea, death (4D’s)
Vitamin B5 (panthothenic acid) – CoA-SH (Fatty acid synthesis)
o Deficiency:
 Rare, dermatitis, hair loss, gastritis
Vitamin B6 (pyridoxal) – amino acid synthesis
o Deficiency:
 Neuropathy, seizures
Vitamin B12 (cobalamin) – homocysteine to methionine
o Deficiency:
 Vegans, pernicious anemia, history of Crohn’s gasterectomy
 B12 deficiency possible with strict vegan with megaloblastic anemia
Vitamin B7 (biotin) – cofactor for carboxylation reactions in gluconeogenesis, Fatty acid
synthesis and metabolism
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Deficiency: Rare
 Dermatitis, gastroenteritis, elevated cholesterol
Vitamin B9 (folic acid) – required for methyl transfers
o Deficiency:
 Neural tube defects (spina bifida, etc)
Vitamin C (ascorbic acid) – serves as a biochemical redox system in the electron transport chain
o Deficiency: scurvy
o Excess: GI disturbances, diarrhea and oxalate kidney stones
B vitamins as coenzymes – Low B’s  increase lactate, alanine
o Pyruvate dehydrogenase (PDH) uses: thiamin, riboflavin, niacin, pantothenic acid,
pyridoxine, lipoic acid, also Mg, P, S, Fe, Zn as cofactors
Minerals – group of inorganic nutrients which are largely considered essential in small amounts
o Absorption is more complex
o Classified as: electrolytes, minerals, trace minerals and ultra-trace minerals
o Electrolytes – Sodium, Potassium, and Chloride are major ions that help establish ion
gradients across cell membranes and maintain water balance
o Calcium and phosphorus – important in skeletal structure; calcium – blood clotting and
hormone function; phosphorus – energy production (ATP)
o Iron – important component of Hgb and many enzymes
o Sulfur – found in connective tissue and functions in metabolism
o Micro minerals (Rizk board review)
 Zinc – cofactor for collagenase; spermatogenesis and growth
 Deficiency:
o Delay in wound healing, decrease in adult hair, hypogonadism,
loss of taste and smell
o Alcohol use predisposes cirrhosis, diabetes mellitus,
acrodermatitis enteropathica
 Copper – cofactor for ferroxidase, attaches iron to transferrin for lysyl oxidase,
cross-linkages in collage and elastic tissue
 Deficiency:
o microcytic anemia, aortic dissection, poor healing, Wilson’s
Disease
o chronic liver disease, basal ganglia degeneration, KayserFleischer ring around cornea
 Selenium – cofactor for glutathione peroxidase (antioxidant); converts peroxide
to water
 Deficiency:
o Weakness and muscle pain, dilated cardiomyopathy
Bioavailability and Absorption impact health status and recommendations
o Enhance absorption of iron with acids
o Inhibit absorption of many minerals with increased dietary fiber
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Micronutrient deficiencies in the US rare but suboptimal intakes common
o Americans: over consume macronutrients
Nutrient assessment
o ABCDE
 Anthropometric
 Biochemical
 Clinical
 Dietary
 Education/Economic (emotional)
o Dietary Reference Intakes (DRI)
 Estimated Average Requirement (EAR)
 Recommended Dietary Allowance (RDA)
 Upper Limit (UL)
 Adequate Intake (AI)
Dietary Supplement Health and Education Act of 1994
o To amend the Federal Food, Drug, and Cosmetic Act to establish standards with respect
to dietary supplements for other purposes
Lecture: 21-22 Metabolism: An Overview
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o
Metabolism – the sum of all the enzyme catalyzed reactions that occur in a living organism
o Catabolism – breakdown of biomolecules to produce energy and the building blocks for
other synthesis (glycolysis)
 Energy released by catabolism conserved in: Nucleoside triphosphates, reduced
coenzymes, and acetyl coenzyme A
o Aerobic vs Anaerobic metabolism
 Aerobic – pathway that require O2 to operate and more specifically lead to ATP
production
 Anaerobic – pathways that operate more specifically lead to ATP production in the
absence of O2
 Glycolysis produces ATP anaerobically, and NADH (requires to be used in
ETC with O2)
 No O2  ETC shuts down  NADH levels rise rapidly  glycolysis inhibited
 During Ischemia, glycolysis kicks in to produce ATP as long as it can
o Anabolism – biosynthesis of more complex molecules from small precursors in reductive
pathways (gluconeogenesis)
o Metabolic pathway – series of coupled reactions in which the product of one reaction acts
as the substrate for the next reaction with regulation of the flow of meatbolites
 Amphibolic pathway – one involved in both anabolic and catabolic processes
 As one pathway is active the other pathway is inhibited (ex. Glycolysis on:
gluconeogenesis off)
Dietary requirements
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Carbohydrates – not essential; can synthesize any carb needed
Essential fats
 Linoleic acid and α-linolenic acid (from dietary plant oils)
 Linoleic acid used to make arachidonic acid (needed for eicosanoid synthesis)
 α-linolenic acid (ALA) used to make eicosapentaenoic acid (EPA) needed to make
doccshexanoic acid (DHA)
o Protein – nine essential amino acids (PVT TIM HaLL = Phenylalanine, Valine, Threanine,
Tryptophan, Isoleucine, Histadine, Lysine, Leucine)
Basal Metabolic Rate (BMR) – energy required to sustain life sometimes called Resting Metabolic
Rate (RMR)
Diet Induced Thermogenesis (DIT) – metabolic rate increase after consuming a meal due to energy
required to digest, absorb, distribute and store fuels
Body Mass Index (BMI) – method for determining whether a person’s weight is in the healthy range
The Fed State
o Fuels we consume are oxidized to meet our immediate energy needs
o Excess fuel is stored as triglyceride and glycogen
o
o
o
Digestion and Absorption – food broken down into smallest units in the mouth, stomach
and small intestine
 Carbohydrate – complex carbs broken down to monosaccharides
 Proteins – broken down to single amino acids
 Fats – emulsified by bile salts forming microdroplets called chylomicrons
Changes in Hormone Levels after a meal
 Glucose in blood stimulates the production of insulin; glucagon levels decrease
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o
o
o
 Insulin stimulates cells to uptake glucose
Fate of Glucose after a meal
 Liver – some immediately metabolized to ATP; remained converted to storage as
glycogen and triglyceride
 Brain and Nervous tissue – highly dependent on glucose as the major energy source
(fatty acids cannot pass the blood-brain barrier); tightly regulated
 Red Blood Cells – glucose only source of energy (no mitochondria); tightly regulated
 Muscle – utilizes glucose from blood for energy and maintain glycogen stores in
muscle; exercise depletes glycogen
 Adipose tissue – insulin greatly stimulates glucose uptake into adipose tissue;
glucose oxidized for energy; glucose provides glycerol moiety for making triglyceride
storage
Fate of Lipoproteins in the fed state
 Chylomicrons – transport triglycerides and cholesterol from intestines to tissue
 Gives muscle (especially heart) first chance to utilize fat; excess fat goes to
adipose
 VLDLs (Very Low Density Lipoproteins) (formed in the liver) transport triglyceride in
the blood to adipose and peripheral tissues
 Triglyceride degraded to free Fatty Acid
 Adipose: fatty acids reformed to triacylglycerol and stored as fat droplets
 Muscle: Fats conjugated with CoA and transported to mitochondira for
oxidation
 Remaining particles cleared by liver or make LDLs (Low density lipoproteins)
Fate of Amino Acids in the fed state
 Most used to synthesize new protein, but some oxidized to yield energy or make
important metabolites (ATP, hormones, neurotransmitters, heme, etc)
 Muscle is a major user of amino acids to make new protein
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o
The Fasted State
o Body utilizes fuel stores to provide energy for tissues
o Strategies to meet energy needs in short term (fasting) different than those used for long
term (starvation)
o
o
o
o
o
o
Blood glucose and the role of the liver during fasting
 Blood glucose levels peak about an hour after eating
 Two hours after eating the body is in the fasting range
 Liver responds to decreased insulin levels, shuts down storage pathways; glycogen
degradation begins
Twelve hours after eating
 Body is in basal state
 Major problem – fatty acids can’t provide carbon for gluconeogenesis, only the
glycerol part of triglycerides can be used as the starting material for
gluconeogenesis
Role of adipose tissue during fasting
 Triglyceride – fatty acids provde the major source of fuel during fasting
 Fatty acid oxidized to acetyl CoA which leads to lots of ATP production
 Liver – most fatty acids taken up used to produce ketone bodies (used by muscle,
kidney and later brain)
Metabolic changes during a short fast
 Liver – maintains blood glucose levels
 Fatty acids from adipocytes provide major source of energy
 Ketone bodies are an important source of energy for many tissues
 Brain continues to rely on glucose
 Byproduct of using amino acids for gluconeogenesis is nitrogen, converted to urea
Metabolic changes during a long fast
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
o
Emphasis shifts away from producing glucose as the major fuel (need for glucose
decreases  less protein degradation  muscle mass spared)
 Muscle decreases use of ketone bodies and uses fatty acid oxidation as primary
source of fuel
 Brain starts to utilize ketone bodies to make acetyl CoA
 Amino acids only used for gluconeogenesis to provide glucose to blood for RBCs
 Less amino acids used for gluconeogenesis, decreased urea product
Role of adipose tissue during a long fast
 Triglycerides of adipose tissue key during prolong fast
 Glycerol used for gluconeogenesis
 Some glucose still used, so protein  amino acids  glucose continues
 Lose fat but also lose muscle (at a slower rate)
Lecture: 23-24 Bioenergetics
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Thermodynamics – collection of laws and principles describing the flows and interchanges of
heat, energy, and matter in systems of interest; three laws describe the majority of
thermodynamic principles
Enthalpy, H – function related to heat transfer and work expenditure in a system
o ∆H refers to the change in enthalpy during a reaction or change in state
 State 1  state 2 (∆H = H2 – H1)
o ∆H reports heat flow in J/mol
o Negative ∆H = heat lost by system; favorable
 Form weak interactions, liberate energy; favorable
o Positive ∆H = heat gained by the system; unfavorable
 Break weak interactions, requires energy; unfavorable
Entropy, S – a measure of disorder
o An ordered state is low entropy
o A disordered state is high entropy
o Increase in entropy is always favored
o ∆S reports change in order
 State 1  state 2 (∆S = S2 – S1)
o Negative ∆S = the system has become more ordered; unfavorable
 Interaction of water with a polar solute, disordered; high entropy
o Positive ∆S = the system has become less ordered; favorable
 Interaction of water with a nonpolar solvent, ordered; low entropy
Free Energy, G – tells how much energy is available for work and allows one to asses reaction
spontaneity (hypothetical quantity)
o G = H- T(S) or ∆G = ∆H - T∆S
o A decrease in free energy is favorable
o ∆G reports reaction spontaneity
o Negative ∆G = the system has decreased free energy
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 Favorable (negative ∆H, large positive ∆S, or both)
o Positive ∆G = the system has increased in free energy
 Unfavorable (positive ∆H, negative or small ∆S, or both)
o If ∆G = 0, reaction at equilibrium (no net change in products and reactants)
o If ∆G < 0, reaction proceeds towards and favors products (spontaneous)
o If ∆G > 0, reaction does not proceed towards products, favors reactants (nonspontaneous)
o Keq = [C][D]/[A][B]
o ∆G = ∆G⁰ + RT ln Keq
 At equilibrium ∆G = 0, therefore ∆G⁰ = -RT ln Keq
 If Keq < 1, ln Keq is negative, ∆G⁰ is positive (products favored)
 If Keq > 1, ln Keq is positive, ∆G⁰ is negative (reactants favored)
o Free energy changes are additive
High energy molecules – contain phosphates and coenzymes
o PEP and 1,3-BPG high; ATP medium; sugar phosphates low
o PEP > 1,3-BPG > ATP > UDP-glucose > acetyl CoA > glucose-6-p
o ATP has high energy because of the phosphate bonds (only a transient carrier)
o Enol Phosphates
 Phosphoenolpyruvate (PEP) has largest free energy of any biomolecule
 Formed from dehydration of 2-phosphoglycerate (2-phosphoglycerate +
enolase (dehydrate)  PEP + ADP  pyruvate + ATP
o High energy phosphates – ATP can transfer energy to enzymes to allow for work or
activate other molecules
 ATP  UDP  UTP + glucose-1-P  UDP-glucose + glycogen  build up
glycogen
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 Energy not coupled (used) given off as heat
Energy transfer in the mitochondrion: Electrons – fuel oxidation gives energy in reducing
equivalents (electrons, NADH/FADH2)
o Electrons from fuel given to NAD+ or FAD to form NADH or FADH2
o Electrons transferred from NADH/FADH2 to the electron transport chain which sets up a
proton gradient
o Energy from proton gradient used to drive ATP synthase to make ATP
Redox Cofactors:
o NAD+/NADH and NADP+/NADPH
o FAD/FADH2 and FMN/FMNH2
o Coenzyme Q
o Lipoic Acid
o Vitamin C
o Tetrahydrobiopterin
Redox Potential
o Electrons transfer from one molecule to another is the Standard Reduction Potential E⁰
o Electrons flow spontaneously from a molecule with a more negative to a more positive
E⁰
Lecture: 24hormonal Regulation of Metabolism

Metabolic Homeostatis – cells need a constant supply of fuels to make ATP
o 190g glucose/day (150g for brain; 40g for other tissues)
o Blood glucose levels of 80-100mg/dL needed to maintain metabolic processes normally
o Balance = metabolic homeostatis
o Strategies for Homeostasis:
 Local control – concentration of metabolites in the blood or cells affects enzyme
activities
 Enzyme velocity depends on [S]: increase S = increase velocity
 Allosteric enzyme activity affected by modulators
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Enzyme isoforms in different tissues have different sensitivities to
metabolites
 Hormonal control – hormones carry messages to individual tissues about the
state of the body and supply of fuel or demand of nutrients
Insulin and Glucagon
o Primary metabolic hormones
o Synthesized and released from clusters of small glands called islets of Langerhans in the
pancreas
 Insulin – produced by β cells
 Glucagon – produced by α cells
o Released in response to circulating levels of fuels in the blood
o Counter regulatory – the effect of each hormone counteracts the effects of the other
 Insulin is the “boss” hormone; glucagon responds
o Insulin – anabolic hormone of the body which signals fed state
 promotes storage of fuels or utilization of fuels for growth
 glucose storages as glycogen in liver and muscle
 conversion of glucose to triglycerides in liver and then storage in
adipose tissue
 amino acid uptake and protein synthesis in skeletal muscle
 increase synthesis proteins required for fatty acid transport and storage
 inhibits fuel mobilization (glycogen breakdown, gluconeogenesis, fatty
acid oxidation)
 Release of insulin is dictated by blood glucose level
 Glucose will stimulate insulin release, then insulin stimulates glucose
uptake
 Insulin level peak about 30-45 minutes following a high carb meal
 Insulin levels return to basal level about 2 hours after meal
o Low glucose will not stimulate insulin release (glucokinase has a
low affinity for glucose; pancreas has GLUT 2: low affinity, high
capacity transporter)
 Insulin: synthesis and secretion
 Insulin is a polypeptide hormone contained in storage vesicles in β-cells
 Release of insulin rapid when glucose enters β-cells
 Insulin is rapidly removed by degradation in liver when blood glucose
levels fall
 Epinephrine decrease release of insulin (fight or flight hormone)
 Signal transduction by insulin – insulin changes target protein phosphorylation
status, which activates or inactivates, depending on the particular protein
 Many effects on the cell – cell growth,
phosphorylation/dephosphorylation cascades for metabolism
 Calcium levels rise with IP3 production
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 Activation of gene transcription
o Glucagon – catabolic hormone of the body which signals the fasted state
 Promotes fuel utilization
 Release of glucose from liver glycogen
 Gluconeogenesis from lactate, glycerol and amino acids
 Mobilizes fatty acids out of adipose tissue  fats go to tissues
 Acts on liver and adipose tissue, not muscle
 Release depends on absence of glucose and insulin (level lowest after a meal)
 Glucagon increases as insulin falls (highest during prolonged fast and in starved
state)
 Glucagon: synthesis and secretion
 Synthesized in the α-cells of the pancreas as pro-glucagon
o Selective proteolytic cleavage to activate glucagon (half-life is
only about 3-5 minutes)
 high protein meals will keep glucagon levels high
 glucagon and other hormones are released to counteract hypoglycemia
 Signal transduction by glucagon
 Operates through cAMP-directed phosphorylation cascade (glucagon
receptor is G-protein coupled and activates adenylate cyclase for
production of cAMP)
 cAMP activates protein kinase A and phosphorylates target proteins
High Carbs vs High Protein
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Insulin vs Glucagon
Glucagon-stimulated phosphorylation: Effect on glycogen
o PKA activation has two major effects on glycogen
 Glycogen synthesis stops
 Glycogen degradation starts
 PKA activates phosphorylase kinase which activates glycogen
phsophorylase
 cAMP turns on glycogen degradation, turns off glycogen synthesis
 Insulin turns on glycogen synthesis, turns off glycogen degradation (insulin
dephosphorylates the proteins that were turned on by phosphorylation)
 Glucagon  phosphorylation  turns off glycogen synthesis, turns on glycogen
breakdown
 Insulin  dephosphorylation  turns on glycogen synthesis, turns off glycogen
breakdown
Other hormones that affect glucose homeostasis
o Central nervous system can also signal hypoglycemia, exercise or other physiological
states
o Epinephrine, norepinephrine, cortisol
o Epinephrine has similar effect to glucagon (increased glycogen degradation)
o
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Lecture: 25-26 The Tricarboxylic Acid Cycle
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Acetyl Coenzyme A
o Most catabolic pathways oxidize fuel sources to the activated portion of Coenzyme A
o CoA has different fates
Function of the TCA cycle
o Hub of anaerobic metabolism responsible for oxidation of acetyl CoA to CO2 and H2O
o Oxidize acetyl CoA to form NADH and FADH2 (used to make ATP by ETC and oxidative
phosphorylation)
o Build up of NADH can stop the TCA cycle from proceeding
Outcome of the TCA cycle – net yield per acetyl CoA molecule (3 NADH, 1 FADH2, 1 GTP)
o During oxidative phosphorylation:
 Gain 2.5 ATP per NADH
 Gain 1.5 ATP per FADH2
 1 GTP
 Produce about 9 ATP molecules per acetyl CoA and 1 GTP

Full turn of the TCA Cycle
o Step 1: condensation of the 4 carbon dicarboxylic acid oxaloacetate (OAA) with the
acetyl group of acetyl CoA forming 6 carbon citrate
o Step 2: isomerization of citrate to isocitrate by aconitase
o Step 3: dehydrogenation of isocitrate from α-ketogluterate (αKG)
 Hydride removed from carbon with –OH and given to NAD+
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Step 4:
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Step 5:
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Results in CO2 loss and formation of double bond
o
CO2 is removed from α-carbon with the addition of Coenzyme A
Two electrons are removed and transferred to NAD+
o
the energy of the succinyl CoA tioester bond is used to generate GTP
Substrate level phosphorylation for the formation of triphosphate (GTP) without
O2
o Step 6: succinate is oxidized to fumarate
 One electron and one proton is removed from each methylene group ad
transferred to FAD (succinate dehydrogenase is also a part of the ETC)
o Step 7: water adds across the double bond of fumarate  malate
o Step 8: OAA is regenerated by the dehydration of malate
 Hydride transferred to NAD+
TCA cycle cofactors
o Requires: TPP, FAD/FMN, NAD+, Coenzyme A, lipoate, Iron, Magnesium, Sulfur, and
Phosphorus
o A boost in metabolism can be seen with an increase in dietary Vitamins B1, B2, B3, B5,
lipoic acid and above minerals
TCA Cycle Energetics: Exothermic
o Three reactions have large negative ∆G⁰ values (reverse reaction is very slow, small
amount of products because products are used in subsequent reactions)
 Formation of citrate
 Formation of αKG
 Formation of succinyl CoA
TCA Cycle Energetics: Endothermic
o Two reactions have positive ∆G⁰ values
 Formation from citrate to isocitrate
 Formation from malate to OAA
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Efflux of intermediates from the TCA cycle – many metabolic pathways intersect the TCA cycle,
and intermediates can be siphoned out of the TCA Cycle
Anaplerotic Reactions – filling reactions provide for TCA intermediates that are depleted by
other pathways (most important is pyruvate + CO2  OAA by pyruvate carboxylase)
o Pyruvate Carboxylase – because of malate accumulation OAA levels can be low or if
acetyl CoA is high, PDH is turned off  pyruvate can be converted directly to OAA which
replenishes the pool and reacts with abundant acetyl CoA to drive the cycle
Pyruvate Dehydrogenase – converts pyruvate into acetyl CoA
o PDH-P is inactive
o Fasting conditions: glucagon high, favors phosphorylated PDH
 Don’t want to use pyruvate to make acetyl CoA, rather use it for glucose
synthesis
Citrate Synthase – large negative ∆G⁰
o Reaction will proceed and be stimulated by acetyl CoA and OAA
o Inhibited by its product – citrate
Isocitrate dehydrogenase – rate limiting step
o Allosterically activated by ADP and Calcium, inhibited by NADH
 Calcium signals “growth” state or energy utilization state
o
o

Malate Dehydrogenase – large positive ∆G⁰
 NADH is a strong stop signal
 NADH causes malate to accumulate
 Excess malate will exit the mitochondrion and enter gluconeogenesis
Lactate Dehydrogenase
o LDH – interconverts pyruvate and lactate using NAD+/NADH
o Tissue dependent – Heart favors pyruvate; RBC favor lactate (RBC have no mitochondria
to use pyruvate)
o Five Isozymes – depends on upon which tissue it is found (ex. B4 favors lactate to
pyruvate in heart; A4 favors pyruvate to lactate in muscle and liver)
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Skeletal Muscle vs Cardiac muscle
o Skeletal muscle does not have much mitochondria so it does not use much pyruvate, so
it is exported out of the cell (glycolysis is a major source of ATP)
o Cardiac Muscle has lots of mitochondria so will use pyruvate and convert lactate to
pyruvate (energy production mainly the TCA cycle)
LDH in the blood above normal levels can give a clue to which tissue is damaged depending on
the isozyme of LDH
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