Module 1: biochemistry and the unit of life

~study of life at the molecular level

~principles of chemistry to explain biology
~study of the molecular logic of life
~all living organisms are remarkably uniform at the molecular level
~All organisms use a common set of building blocks to create common categories of biomolecules
(nucleic acids, proteins, polysaccharides, lipids)
~Foundations of life chemical, energy, genetic and evolutionary.
~Simple and common ingredients of life (carbon, oxygen, hydrogen and nitrogen) and account for go of
most organisms
~all known life forms are made of carb on
~chemical elements within the biosphere are carbon, oxygen and hydrogen
~air contains inert form of N2, Nitrogen within biosphere comes from plants
~carbon has the ability to make 4 types of linkages
~silicon is the next best candidate as a chemical foundation for life, also can form four covalent bonds,
highly abundant in earth's crust
~carbon-to-carbon stronger, more energy released on combustion (carbon more solvable and recycle in
the biosphere than silicon to Silicon
~functional groups; unique based on size, shape, charge, reactivity and hydrogen bond capacity
↳ determine function, structure and properties of biomolecules
~Structure of biomolecules dictates function
~conformation - flexible spatial arrangement of atoms within a molecule
configuration - fixed spatial arrangement of atoms within a molecule (double bonds or chiral centers)
Geometric (cis-trans) isomers same chemical formula but differ in configuration can have very different
biological properties of groups with respect to non-rotating double bond
~Cis- "on this side" ~trans- "across”
~as soon as double bonds are in place, the have more limits
~all amino acids have amino group, carboxyl group, hydrogen and side chain
~construction- Biomolecules often constructed exclusively from one stereoisomer
~Interactions -Interactions between biomolecules, as well as between biomolecules and small molecules
~L/D versions we use (versions in all amino acids, we don't use D versions because they are pictures of
what should be happening and cannot be used
~Synthesis of chemical bonds which have asymmetric carbons result in a mixture of all the chiral forms a
form may have different biological activities
~Biomolecules often polymers of simple building blocks
~Structure and function of resulting biomolecules are more complex than their precursor molecules,
greater than the sum of parts
- Simplicity- simple and conserved reactions for synthesis and degradation
- Recycling- biomolecules can be digested back to component building blocks which are reusable
- Diversity- incredibly complex molecules can be generated
- Four major classes of biomolecules Proteins, carbohydrates, nucleic acid and lipids
PROTEINS
- Linear polymers of amino acids
- Amino acids link together to form linear chains that fold into complex patterns with district
biological activities (sequence-> structure-> function)
CARBOHYDRATES
- Polysaccharides- link together with glycosidic linkages
- Monosaccharides- link together to form linear or branched polymers
- Role of high order polysaccharides serve as structural, energy storage and cellular recognition
NUCLEIC ACID
- Linear polymers of nucleotide building blocks (5 blocks of DNA and RNA)
 - Roles- storage and utilization of genetic information
LIPIDS
- Lipids are linked by hydrophobic drive, they are not linked through covalent linkages
- Role- energy storage, formation of membranes and signalling
PROKARYOTES
- Small, single celled organisms
- Rapid growth, quick adaptation
EUKARYOTES
- Large complex organisms, multicellular
- Organelles to support functions
CELLULAR FOUNDATION
- Undergo complex, organized interactions within the cell
- In vitro (glass)- study behaviour of molecules outside of the context of the cell and organisms
- In vivo (living)- study behaviour of molecules occurring within the complexity of the cell or
organism
ENERGY FOUNDATION
- First law of thermodynamics- total amount of energy in universe remains constant although
energy forms may change
- Cells are highly effective transducers of energy
- Second law of thermodynamics- total entropy of the universe is continually increasing
- Living systems and their biomolecules require a high degree of organization
- Enthalpy(H)- number and kind of bonds
- Entropy(S)- degree of randomness
- Temperature- degrees in kelvin
- Endergonic(G>0) nonspontaneous process, input of energy to proceed
- Exergonic(G<0) spontaneous (without energy input) release free energy
- Thermodynamically unfavorable reactions by coupling energy requiring to energy releasing
reactions
- Sum of energy changes is negative, overall process is exergonic
- Catabolic- breaking things down
- Anabolic- building things up
- Energy exerted to the form of adenosine triphosphate (ATP) serves as energy currency
- ATP serves as the link between catabolic and anabolic reactions
GENETIC FOUNDATIONS
- Perpetuation of life requires that genetic information: stored in a stable form, expressed
accurately in the form of gene products, reproduced with minimal errors
- Deoxyribonucleic acid (DNA) provides instructions for forming all other cellular components,
template for production of identical DNA molecules to be distributed to the progeny when a cell
divide
- DNA has two complimentary strands
- Each strand is linear polymer of four different types of building blocs
- Linear sequence within the strands that encodes information
- Nucleotide sequence of genes dictates the sequence of amino acids incorporated into
corresponding protein
- Amino acid sequence of the protein dictate’s structure, structure dictates biological activity
EVOLUTIONARY FOUNDATION
- Random changes in genotype result in change in phenotype

Modules two: Water

GENERAL
- Most abundant molecule in living organisms
 - Both passive and active roles in biochemistry
- Passive; the structure of biomolecules form in response to interactions with water
- Active; water is a participant in many biochemical reactions
STRUCTURE/FUNCTION
- Oxygen and hydrogen differ in their electronegative
- Oxygen is more electronegative than hydrogen
- Water has permanent dipole
- Oxygen has partial negative charge and each hydrogen has partial positive charge
- Water dipole-> form electrostatic interactions with charged molecules which form hydrogen
bonds
GENERAL
- Hydrogen bonds-> electrostatic interactions between an electronegative atom with hydrogen
covalently linked to another electronegative atom with a free electron pair
- Oxygen and nitrogen are common hydrogen bonders with biomolecules
- Oxygen and hydrogen can serve as hydrogen bond donors and acceptors
HYDROGEN BONDS STRENGTH AND GEOMETRY
- Relatively weak, 5% the strength of covalent bond
- Double the length of covalent bonds
- Strength depends on geometry
- Anti-parallel beta sheets are more stable than parallel because geometry of hydrogen bonding
UNUSUAL PROPERTIES OF WATER
- Each water molecule can donate and accept two hydrogen bonds
- Each molecule has potential to participate in four other water molecules
- Flickering clusters average at 3.4 hydrogen bond
- Hydrogen bond between water molecules confer great internal cohesion, influences the properties
of water
- Heat of vaporization; amount of heat requires to vaporize a liquid at its boiling temperature
- Specific heat capacity: amount of heat required to raise the temperature of a substance one degree
- Water has a higher melting point, boiling point and heat vaporization
- Ice has a lower density than liquid water therefore ice floats
POLYWATER
- Forced through quartz tubes
- Higher boiling point, lower freezing point, higher viscosity
- Within polywater was self-propagating and could be used as a weapon
ELECTROSTATIC INTERACTIONS
- Water molecules can interact, dissolve
- Charged solutes through formation of layers of hydration
- Water molecules have small size, permanent dipole, this creates a versatility with
positive/negative ions
HYDROGEN BONDS
- Biomolecules have functional groups that form hydrogen bonds
- Functional groups can create hydrogen bonds within the same molecule, other biomolecules or
water
- Small size and ability to be a donor or an acceptor
SOLUABLILITY OF DISSOLVED MOLECULES
- Solubility of molecules depend on the ability of interactions with water molecules
- Molecules that carry positive/negative charge or are hydrogen acceptors/donors have greatest
water solubility
- Hydrophilic- loves water (polar)
- Hydrophilic- hates water (nonpolar)
- Amphipathic molecules have hydrophobic and hydrophilic portions
 - Many important gasses are not as soluble in water or blood
- Limited solubility creates challenge for transport
- Specialized transport proteins and strategies are required for CO2 and O2 transport
BEHAVIOUR OF AMPHIPATHIC SUBSTANCES
- Amphipathic molecule is mixed with hydrophilic regions that interact with water by hydrophobic
regions cluster together to present the smallest surface
- Forces that hold nonpolar regions of molecules called hydrophilic interactions
- Most biomolecules are amphipathic
- Hydrophobic drive is primary driving force in formation and stabilization of biomolecular
structures
CRUCIAL TO MOLECULAR STRUCTURE AND FUNCTION
- Biomolecules represent stable polymers of covalently linked building blocks
- 3D structures formed by these polymers are made through noncovalent interactions
- Interactions between biomolecules is also determined by noncovalent interactions
- Noncovalent interactions enable the formation and stabilization of structures of biomolecules,
recognition/interactions between biomolecules, binding of reactants to enzymes
- Noncovalent interactions within the biomolecules: hydrogen bonds, ionic (electrostatic)
interactions, hydrophobic interactions and Van Der Waals interactions
HYDROGEN BONDS
- Many functional groups with biomolecules have hydrogen bonding capacity
- Groups can form hydrogen bonds with water molecules, groups with same molecule (intra),
groups with other molecules (inter)
- Hydrogen bonds are critical for specify biomolecular interactions, not the formation
- Unfolded state, groups can hydrogen bond with water
IONIC (ELECTROSTATIC) INTERACTIONS
- Electrostatic interactions between charged groups can be attractive or repulsive
- Magnitude of contributions of ionic interactions to biomolecular structures is reduced by
shielding of these groups by water molecules
- Water shields the charge groups, greatly diminishing the strength of the interaction
- Strength of electrostatic interactions depend on the distance separating the atom
VAN DER WAALS FORCES
- Interactions between permanent and induced dipole, short range and low magnitude interactions
- Two atoms are separated by sum of the Van Der Waals radii, attraction is maximal
- Two surfaces of complimentary shapes come together a large number of atoms are brought into
Van Der Waals contact
- Every atom has a “personal space” they like to be respected
HYDROPHOBIC EFFECT
- drive to have polar interacting with water and nonpolar regions shielded away from water
- in protein folding: nonpolar side chains cluster in the interior of the protein away from water,
polar and charged side chains remain on the outer surface facing water
- folding of protein involves creation of a more ordered state, contradicts the second law of
thermodynamics
THERMODYNAMIC OF THE HYDROPHOBIC EFFECT
- water molecules around hydrophobic molecules are more ordered than they would be pure water,
introduction of the nonpolar molecule causes a decrease in the entropy of water
- nonpolar molecules or regions releases some of the ordered water molecules, results in an
increase of the entropy of water
- folding of a polypeptide decreases the entropy of the polypeptide but increases the entropy of
associated water
IONIZATION OF WATER
- water has limited tendency to ionize hydrogen ions (HA) and hydroxide ions (OH-)
 - kw is product of water
PH SCALE
- more convenient to express (14) as pH
- pH is a log scale such that the difference of 1 pH unit equals a lo fold difference (H+)
WEAK ACID AND BASES HAVE CHARACTERISTIC DISSOCIATION CONSTANTS
- strong acids and bases dissociate completely in water
- weak acids and bases do not associate completely in water
- Ka values often expressed as pKa's (pKa = -log Ka
TITRATION CURVES REVEAL THE PKA OF WEAK ACIDS
- ratio of acid to conjugate base changes over the course of the titration curve
- when pH = pKa then A = HA
- when pH= pKa the solution is best able to resist changes in pH
- buffering regions extend to one unit above and one unit below the pKa point
- pH < pKa more pronated form
- pH> pKa more unpronated form
BUFFERS ARE IMPORTANT TO BIOLOGICAL SYSTEMS
- need to maintain constant pH
- changes in pH could alter the protonation state of biomolecules, this could change the
- structure and function
- number of weak acids that serve to buffer biological systems
- compensatory respiratory alkalosis serves to maintain the ratio of H2COs/HC05 to maintain
constant pH
- isothemic maintain pH heavy systems of weak acids in the body to be able to function as buffers

module 3: amino acids

GENERAL
- amino acids are building blocks of protein
- strands fold onto each other to form bioactive 3D conformation
- Proteins are linear polymers of amino acids
- all proteins are produced from zo strand amino acid
- living organism use the same pool of amino acids to build their proteins
PROTEINS ARE POLYMERS OF AMINO ACIDS
- distinct advantages of creating biomolecules as polymers of smaller, simpler building blocks
- 1. simplicity of chemistry: one type of reaction of polymerization, a second type of reaction for
degration
- 2. Recycling: biomolecules can be digested back in component building blocks which are
reusable for production
- of other biomolecules
- 3. Diversity: the vast number of molecules of varying lengths and sequences
GENERAL STRUCTURE
- common amino acid feature. hydrogen, side chain group, Carboxyl group, Central Alpha Carbon
and amino groups
- the 20 amino acids differ side chains (R) group
CHIRALITY
- glycerine, does not have the alpha carbon is bonded to four different groups which creates a
- four different groups occupy unique spatial arrangements giving different stereoisomers (L/D
isomers)
- Biologically proteins are made almost exclusively from L isomers
THOUGHTS OF GROUPINGS
- textbook amino acids based on their side chains groups include Nonpolar aliphatic, aromatic,
polar uncharged, polar positively charged and polar negative charge
 - effective way to sub divide amino acids
NONPOLAR
- mainly hydrocarbon side chains
- residues with nonpolar chains are often buried in the core of the proteins
- Proline often found at polypeptide turns, usually combination with glycerine
- glycine is the smallest amino acids and is the only one which is not chiral
- methionine is one of two amino acids with a sulfur group within its side chain
AROMATICS
- histidine can also be considered as an aromatic
- tyrosine can be post translation modified through phosphorylation
- phosphorylation is a mechanism to regulate protein function
- other amino acids with hydroxyl groups (serine and threonine) also can be phosphorylated
- tryptophan, a precursor or serotonin, became a popular supplement in the 1980s
POST TRANSLATION MODIFICATION
- some amino acids can be covalently modified after their incorporation into a protein
- phosphorylation is an example of post translational modification
- phosphoryl groups are added by kinases to specific, hydroxyl group containing amino acids (tyr,
ser, thr)
- modifications are reversible
- just because a protein is present, doesn't mean it is active
- Kinase →phosphorylate
- phosphatase - removes phosphoryl groups
POLAR UNCHARGED
- serine and threonine can undergo phosphorylation of their hydroxyl groups
- two cysteines can form a covalent linkage called disulfide bonds
- disulfide bonds are important covalent linkages for stabilization of some protein structures
- more polar than the other ones but not as polar as the ones that are charged
- potential to form hydrogen bonds from their side chains
DISULFIDE BONDS
- form through oxidation of the sulfhydryl groups of two cysteine residues to form a covalent
linkage
- disulfides stabilize protein structures
- cysteine residues forming a disulfide bond must be proximal in space within the protein structure
- disulfide bonds can be inter or intramolecular
POSITIVELY CHARGED
- lys and Arg always carry a +! charge at physiological pH
- histidine's imidazole group has a pka near physiological pH such as a fraction of cellular histidine
will be 11 and the rest o
- enzymatic reactions histidine serves as proton donor/acceptor
NEGATIVELY CHARGED
- aspartate and glutamate carry a net charge of - a physiological pH
- glutamate is responsible for one of the five basic tastes umami, used as a flavor enhancer
ACID/BASE PROPERTIES
- every amino acid has at least two groups that accept and donate protons (diprotic)
- all amino acids have the alpha carbon carboxyl group and amino acid group
- triprotic amino acids have ionizable groups in their side chains
- diprotic have two buffering regions
- triprotic have three buffering regions
- ionizable groups in the amino acid groups: Carboxyl groups, amino groups, side chains of the
triprotic amino acids
- ionizable groups have specific pKa. the pH at which that group changes its pronation state
 - when pH is below the pka, the protonated form predominates (HAL
- when pH is above the pKa, the unprotonated form predominates (A
- dipolar ion of an amino acid is called zwitterion
TITRATION CURVES OF CARBOXYL AND AMINO ACIDS
- all amino acids have both carboxyl (pKa 2.0) and an amino acid (pKa 10.0) groups
- pH 7.4 these groups will be in the coo- and NH3+ forms
TITRATION OF GLYCINE
- isoelectric point of an amino acid is the pH at which the net charge on the molecule is equal to
zero
- isoelectric point is the average of the pKas on either side of where the net charge is equal to zero
- all diprotic would have similar titration curves and iso electric points
TITRATION OF GLUTAMATE
- titration curves and isoelectric points of aspartate and glutamate would be quite similar
TITRATION OF HISTIDINE
- pK1 = 1.82 pKr=6.0 pK2 = 9.17

module 4: three-dimensional structure of proteins

PEPTIDE BONDS: GENERAL
- peptide bonds are covalent linkages between amino acids
- form by condensation reactions involving loss of a water molecule
- formation of a peptide bond eliminates the a carboxyl and a amino charged groups (important for
protein folding)
- peptide bonds are independent of the amino acids being joined
- residues - what is left behind when the water leaves (amino acids)
PEPTIDE BONDS: POLYPEPTIDE AIN CHAINS
- conserved nature of peptide bonds, repeating pattern within the main chain
- main chain is the constant portion of the polypeptide, side chains are variable
- main chain is repeating pattern NCCNC
PEPTIDE BONDS: PARTIAL DOUBLE BOND CHARACTERISTIC
- rotation around C-N peptide bond is restricted due to partial double bond characteristics
(resonance structure)
- consequence of partial double bond characteristic the six atoms of the peptide group rigid and
planar
PEPTIDE BOND: CONFIGURATION
- partial double bond of the particle bond creates cis-trans isomers
- Oxygen of carbonyl group and the hydrogen of the amide nitrogen are usually trans to each other
- trans configuration is favored as the is configuration is more likely to cause steric interference
between side chain groups
- steric exclusion means two groups can't occupy same space at the same time
PROTEINS: FOUR LEVELS OF PROTEIN STRUCTURES
- Primary structure linear sequence of amino acids (what is grouped to what)
- secondary structure localized interactions within a polypeptides (folding)
- tertiary structures final protein folding pattern of a single polypeptide (final folding structure,
most proteins only go up this high)
- quaternary structures folding pattern when multiple polypeptides are involved (final step if more
than one polypeptide is involved
PRIMARY STRUCTURE: GENERAL
- defines linear arrangement of amino acids in a polypeptide
- Primary structure is presented from N terminus (amino) to the c(carboxyl) terminus
- when looking at a polypeptide or protein at one end there will always be amino group at the other
end always be a free carboxyl group
 - info specifying correct folding pattern
- Primary structure is often determined through investigation of the corresponding gene
SECONDARY STRUCTURE: GENERAL
- represents localized patterns of folding in a polypeptide
- maintained by hydrogen bonds between main chain amide and carbonyl group
SECONDARY STRUCTURE: CONSERVED ACROSS PROTEINS
- elements of secondary structure are found in different proteins
- retain overall characteristics independent of proteins
SECONDARY STRUCTURE: TWO KEY RULE
- optimize the hydrogen bonding potential of main chain carbonyl and amide group
- represents a favored conformation of the polypeptide chain
SECONDARY SRUCTURE: MAIN CHAIN HYDROGEN BONDING GROUPS
- each peptide bond has a hydrogen bond donor and acceptor group
- equal number of hydrogen bond donors and acceptors within the polypeptide main chain
- optimizing hydrogen bonds
SECONDARY STRUCTUIRE: CONFORMATION OF THE POLYPEPTIDE CHAIN
- each a carbon is held within main chain through single bonds which has freedom of rotation
- bonds defined as Phi Psi
- steric interference prevents the information of most conformations
- Ramachandran plots illustrate the possible combination of phi and psi
- Phi/psi combination are observed in proteins are highlighted
- favored conformation correspond to common element of secondary structures
ALPHA HELIX: HYDROGEN BONDS
- right-handed helix with 3.4 residues/turn
- stabilized by hydrogen bonds which run parallel to the axis of the helix
- carbonyl groups point toward the C terminus
- amide group to the N terminus
- each carbonyl of residue n hydrogen bonds with amide group of residue n + 4
ALPHA HELIX: AMINO ACID SEQUENCE AFFECTS STABILITY
- most sequences can theoretically form a alpha helix there are guidelines and trends
- proline →rigity, is not usually found in a helices
- glycine→ flexibility, also uncommon in a helices
- amino acids with sidechain branches are less common due to steric interference
- amino acids with hydrogen bonding groups near the main chain are also less common
- charged residues tend to be positioned to form favorable ion pairs
ALPHA HELIX: THE HELIX DIPOLE
- every peptide bond has a small electrical dipole
- dipole communicated through helix by a hydrogen bonding giving the helix has a net dipole
charge:
↳ N terminus has partial positive dipole charge
↳ C terminus has partial negative dipole charge
- dipole is stabilized by residues at each termini whose charge oppose the helix dipole
↳ negatively charged residues at the N terminus
↳ positively charged residues at the C terminus
ALPHA HELIX: AMPHIPATHIC HELICIES
- residues separated by three or four positioned in the primary sequence will be on the same side of
a alpha helix
- residues separated by two residues in the primary structure will be on opposite sides of the helix
- positioning of hydrophobic and hydrophilic residues within the primary structure generates an
amphipathic helix with
- polar and nonpolar faces
BETA SHEETS: GENERAL
- beta sheets involve multiple & strands arranged side by side
- beta sheets are made up of beta strands
- beta sheets often involve 4/5 strands
- conformation: fully extended polypeptide chains
- hydrogen bonding pattern: stabilized by hydrogen bonds between co and NH on adjacent strands
BETA SHEETS: PARALLEL AND ANTIPARALLEL
- parallel beta sheets the strands run in same direction
- antiparallel beta sheets the strands run in opposite direction
- antiparallel beta sheets are more stable due to better geometry of hydrogen bonding
BETA SHEET: MIXED BETA SHEETS
- beta sheets can be parallel, antiparallel or mixed
- mixed beta sheets contain both parallel and antiparallel beta strands
BETA SHEETS: AMPHIPATHIC BETA SHEETS
- side chains tend to alternate above and below the polypeptide chain
- alternate polar and nonpolar residues within the primary structure of a beta sheet will result in an
amphipathic beta sheet
PROTEINS: TERTIARY STRUCTURE
- final folding pattern of a single polypeptide
- biological active folding pattern is the native conformation
- amino acid sequence determines tertiary structure
- long range aspects of sequence interactions within a polypeptide
- residues separated by great distance in primary structure may be in proximity in tertiary structure
- different proteins have different tertiary structures which relates to their unique functions
- different proteins vary in their content of alpha helices and beta sheets
PROTEINS: CONFORMATION IS STABILIZED BY WEAK INTERACTIONS
- final folding pattern of a single polypeptide
- biological active folding pattern is the native conformation
- amino acid sequence determines tertiary structure
- long range aspects of sequence interactions within a polypeptide
- residues separated by great distance in primary structure may be in close proximity in tertiary
structure
- different proteins have different tertiary structures which relates to their unique functions
- different proteins vary in their content of alpha helices and beta sheets
PROTEIN: FOLDING
- folded proteins occupy low energy state of greatest stability
- low energy state may only marginally stable
- protein folding is a rapid process indicating proteins don’t sample all possible folding patterns
- protein folding can be imagined as a funnel where many unstable conformations collapse to a
single, stable folding pattern
- proteins spontaneously fold to their native conformations
PROTEIN: DENATURATION
- denaturation: disruption of native conformation with loss of biological activity
- energy required for denaturation is often small, perhaps only a few hydrogen bonds
- protein folding and denaturation is a cooperative process
- denaturation is reversible
PROTEIN: QUARTERNARY STRUCTURE
- multiple subunits in which each subunit is a separate polypeptide
- may involve multiple subunits of the same polypeptide or different polypeptides
- subunits usually associate through noncovalent interactions
- reserved for proteins of more complex biological function
 - may help stabilize subunits and prolong life of protein
- unique active sites produced at the interfaces between subunits
- facilitate unique and dynamic combinations of structures/functions through physiological changes
in tertiary and quaternary structure (hemoglobin)
- conversation of functional subunits more efficient than selection for new protein with ideal
function
PROTEIN: STRUCTURE AND FUNCTION
- biological roles of proteins include enzymes, storage and transport, physical cell support and
shape, mechanical movement,
- decoding cell information, hormones / hormone receptor, many other specialized functions
- diversity of function enabled by diversity of structure
- proteins show extreme structural and functional diversity
PROTEIN: NUMBER AND DIVERSITY
- different organisms have different numbers of proteins
- represents the minimum number of proteins, additional isoforms are generated through post
translational modifications
PROTEIN: SIZE
- proteins are loo to loo amino acids in length
- at 51 amino acids, insulin is threshold of when polypeptides become a protein
- largest protein discovered is titin with an isoform of 34350 amino acids
- umber of amino acids in a protein is approx. by dividing the proteins molecular weight by lo
FIVE PROTEIN FACTS
- function of a protein depends on structure
- three-Dimensional structure of a protein is determined by its amino acid sequence
- noncovalent forces are most important forces stabilizing protein structure
- huge number of unique protein structures, there are common structural patterns
- isolated protein usually exists in one, or small number of structural forms
PROTEIN: STRUCTURE/FUNCTION EXAMPLES
- Fibrous proteins. Keratin, collagen, silk
- Globular proteins: myoglobin, hemoglobin
KERATIN: PRIMARY AND SECONDARY STRUCTURES
- Keratin is the principal component of hair, wool, horns and nails
- primary structure of Keratin has pseudo seven repeat where position a and d are hydrophobic
residues
- secondary structure Keratin forms alpha helix
- residues from position a and d end up on the same face of the helix resulting in hydrophobic strip
along length of the helix
KERATIN: COILED COILS
- two amphipathic helices of keratin interact to bury their hydrophobic faces together
- coiled coils are formed when two or more helices entwine to form a stable structure
- coiled coil keratin involves two right-handed helices wrapping around each other in left-handed
fashion
KERATIN: POST TRANSLATIONAL STABILIZATION
- strength of Keratin arises from covalent linkages of individual units into higher order structures
- individual units are linked together through disulfide bonds
- disulfide bonding will determine the strength of the overall structure
COLLAGEN: PRIMARY AND SECONDARY STRUCTURE
- collagen is a major protein of vertebrates
- primary structure, collagen contains repeat of Gly-X-Y where x is often proline
- secondary structure, collagen forms a left-handed helix of three residues per turn
COLLAGEN: COILED COIL
 - three left-handed helices of collagen come together to form a coiled coil
- three left-handed helices wrap around each other in right handed fashion
- bulky side chains of proline are on the outside of the coiled coil
- small side chains of the glycerine residues are in the tightly packed core of the coiled coil
COLLAGEN: POST TRANSLATIONAL MODIFICATION
- strength of collagen arises from covalent linkages between individual units into higher order
structures
- these linkages occur from residues that undergo post translational modification (hydroxyproline,
hydroxylysine)
- crosslinks occur with age, accounting for the increasing brittle character of aging connective
tissue and tougher meat
- covalent crosslinks of collagen involve post translationally modified residues
- enzymes performing these modifications require vitamin C
- without modified residues, collagen cannot form stabilizing crosslinks
COLLAGEN: SCURVY
- vitamin C deficiency scurvy leads to weakened structures of collagen with manifests in Skin
lesions, fragile blood vessels, bleeding gums, bruises, tooth loss, poor wound healing, bone pain,
eventual heart failure
- demonstrates that citrus prevents cures scurvy was one of the first controlled human clinical trials
COLLAGEN: GENETIC DISEASE
- genetic disorders involving collagen and related connective tissue
- some diseases can associate with brittle and abnormal bone structure, weakened cardiovascular
capabilities loose skin and joints and hyper flexibility
SILK: PRIMARY AND SECONDARY STRUCTURE
- silk fibroin is produced by insects and spiders for formation of webs and cocoons webs and
cocoons need flexibility and strength
- primary structure, most silk has six residue repeats
- secondary structure, silk composed from beta sheets
- fully extended polypeptides of offer considerable strength
- cross sectional basis silk is one of the strongest known materials
SILK: FLEXIBILITY AND STRENGTH
- appreciate molecular basis of strength and flexibility of silk, one needs to consider its structure in
each dimension
- fully extended polypeptide chains (strength)
- association of strands by hydrogen bonding (flexible
- association of sheets by Vander Waals and hydrophobic interactions (flexible)
SILK: MEDICAL APPLICATIONS AND GENETIC ENGINEERING
- enticing properties, spider silk has enormous potential for medical applications
- exciting properties of silk are matched by challenges of its availability
PRIONS: NEW FORM OF INFECTIOUS DISEASE
- prion is a novel paradigm of infectious disease based on misfolding of a self protein into a
pathological, infectious information
- Prions is fatal, untreatable neurodegenerative disease
PRIONS: DISEASE SPECIFIC VACCINES
- protein misfold, new regions are exposed for antibody binding
- misfolding dependent epitopes are termed disease specific epitopes (DSEs)
- DSEs appear ideal vaccine targets
- antibodies induced against PSEs only bid unhealthy form of the protein sparing the function of
healthy form

Module 5: myoglobin and hemoglobin

PROTEIN FUNCTION: LIGANDS
- Proteins undergo reversible interactions with other molecules
- interactions can serve to regulate protein function
- molecule reversible bound by protein is called ligand
- ligand can be any kind of molecules, including another protein
PROTEIN LIGANDS: SPECIFICITY
- Ligand binds at a specific site on the protein called binding site
- binding site is usually complimentary to ligand in terms of shape, charge, hyperchromicity and
hydrogen potential
- protein may have multiple binding sites of multiple ligands
PROTEIN LIGANDS: INDUCED FIT
- binding of a ligand may cause conformational change in the protein
- induced fit can change the properties of the protein
- changes in protein structure often relates to changes in function
OXYGEN DELIVERY AND STORAGE: OVERVIEW
- every cell requires a constant supply of oxygen
- multicellular organisms, solvability of oxygen is too low to meet oxygen requirements through
passive diffusion
- amino acid side chains not well suited for reversible binding of oxygen
- transition state metals have strong tendency to bind oxygen but produce damaging free radicals
- proteins for oxygen storage and delivery
- heme groups to safely harness irons oxygen binding properties
OXYGEN AS A LIMITED RESOURCE
- myoglobin and hemoglobin serve distinct complimentary and physiological roles
- myoglobin and hemoglobin share many structural and functional features
- myoglobin (Mb) ~ monomeric protein facilitates oxygen storage in peripheral tissue
- hemoglobin (Hb)~ tetrameric protein found in red blood cells that transports oxygen from lungs
to periphery
REVRSIBLE OXYGEN BINDING: HEME PROTHETIC GROUP
- Oxygen is poorly solvable in aqueous solutions
- multicellular organisms depend on the evolution of proteins that could transport and store oxygen
- amount of available oxygen which can be delivered within the organism can limit its side
- Insects grown in the presence of elevated oxygen can achieve greater sizes
HEME PROSTHETIC GROUPS: CARBON MONOXIDE POISIONING
- cellular iron is bound in forms that sequester it makes it less reactive
- heme consists of protoporphyrin ring system bound to a single iron atom
- iron2+ binds to oxygen iron3+ cannot bind to oxygen
- ring system provides four coordinating interactions with the iron atom
- myoglobin and hemoglobin both use heme
- heme is bound with discrete pockets of myoglobin and hemoglobin
- iron24 seeks six coordinating interactions:
o four come from interactions with heme
o fifth comes interaction with an imidazole group of a proximal histidine residue
o sixth position is for oxygen binding
- distal histidine provides a stabilizing interaction for bound oxygen
OXYGEN BINDING PROTEINS: MYOGLOBIN VS HEMOGLOBIN
- carbon monoxide has a similar molecular structure as oxygen
- carbon exerts its deadly effects by competing with oxygen for binding to heme
- carbon monoxide binds heme with zoo times greater affinity than oxygen
OXYGEN BINDING PROTEINS: MYOGLOBIN VS HEMOGLOBIN FUNCTIONS
- myoglobin, single subunit tertiary structure)
 - with a single heme group myoglobin can bind one oxygen molecule
- hemoglobin, 4 subunits (quaternary structure)
- four heme groups, hemoglobin can bind four oxygen molecules
- each subunit of hemoglobin closely resembles myoglobin
MYOGLOBIN: STRUCTURE
- myoglobin has a higher affinity for oxygen than hemoglobin
- myoglobin has a hyperbolic curve of oxygen binding
- binding of oxygen by hemoglobin displays sigmoidal behaviour, indicates cooperativity of
oxygen binding
MYOGLOBIN: OXYGEN SATURATION CURVE
- small globular protein consisting of:
o a single polypeptide of 153 residues arranged in eight alpha helices
o a heme (iron porphyrin) prosthetic group
- sperm whale myoglobin was the first protein structure determined by x-ray crystallography
MYOGLOBIN: FRACTION SATURATION
- Oxygen saturation curve of myoglobin is hyperbolic, indicating a single oxygen binding constant
- amount of oxygen required to half saturate the protein is qualified by P5o (P50 of myoglobic is 3
torr)
- pO2 in lungs is typically 100 for and 20 for in periphery
OXYGEN TRANSPORT IN THE BLOOD: HEMOGLOBIN
- partial pressure of oxygen is calculated by theta = p02/P02 + P50
- peripheral tissues, partial pressure of oxygen is 20 four
- lungs, partial pressure of oxygen is you found
- myoglobin has a very high affinity for oxygen and is normally nearby saturated with oxygen
everywhere in the body
ALLOSTERIC PROTEINS: GENERAL
- allosteric is derived from Greek word Allos (other) and stereos structural allosteric: other
structure
- allosteric proteins have T(inactive) and R(active) forms
- T and R forms are in rapid equilibrium
ALLOSTERIC PROTEINS: HEMOGLOBIN
- protein that binds oxygen with high constant affinity would saturate with oxygen in lungs but not
release it to tissues
- protein with lower oxygen affinity would be able to release oxygen to tissues would not have
sufficient affinity to saturate in lungs
- hemoglobin solves the problem by undergoing transitions from high to low affinity states
- proteins with a single ligand binding site cannot achieve this cooperative effect
ALLOSTERIC PROTEINS: MODULATORS (EFFECTORS)
- allosteric effectors (modulators) bind allosteric proteins at a specific site
- allosteric effectors can be either can be either activators or inhibitors
- allosteric activators stabilize the R State: allosteric inhibitors stabilize the T state
- normal ligand and modulator are the same, the interaction is homotropic
- modulator is different from the normal ligand the interaction is heterotopic
ALLOSTERIC PROPERTIES OF HEMOGLOBIN
- binding and release of oxygen from hemoglobin are allosterically regulated
- binding the first oxygen by hemoglobin causes conformational change making change making it
easier to bind subsequent oxygen
- Oxygen binding promotes and stabilizes the R State of hemoglobin which has higher oxygen
affinity than the T state
ALLOSTERIC PROPERTIES OF HEMOGLOBIN: T TO R TRANSITION
- T state hemoglobin, iron atom is just outside the plane of the heme ring
 - transition to R State (oxygen bound) iron moves into plane of the ring
- minor movement within one subunit causes structural changes that translate to quaternary
structure of the protein
HEMOGLOBIN: OXYGEN SATURATION CURVE
- critical to look at changes in saturation of hemoglobin over the physiological range oxygen
- partial pressure of oxygen found in the lungs, hemoglobin completely saturates with oxygen
- partial pressure of oxygen found in periphery, hemoglobin releases over half of its oxygen load
- P50 of hemoglobin closely matches partial pressure of oxygen found in periphery
- hemoglobin is most sensitive for oxygen release at partial pressure of oxygen found in the
periphery
- allows hemoglobin to sense and respond to changes in oxygen levels in regions at greatest risk of
hypoxia
ALLOSTERIC INHIBITION OF HEMOGLOBIN: 2,3 BISPHOSPHO-D-GLYCERATE
- highly purified hemoglobin indicated by an extremely high affinity for oxygen
- limit the ability of the ability of the protein to release oxygen to the periphery
- replacing various components of blood revealed that 2,3 Bisphosphoglycerate decreased
hemoglobin’s' affinity for oxygen
- 2.3 Bisphosphoglycerate is a heterotopic allosteric inhibitor of hemoglobin
- 2.3 BPG carries five units of negative charge
- pocket formed at the interface between subunits of deoxyhemoglobin contains six positively
charged residues
- pocket is unique to deoxyhemoglobin
FETAL HEMOGLOBIN: 2,3 BISPHOSPHO-D-GLYCERATE
- fetus "breaths" in the womb by stripping Oxygen away from maternal blood
- Fetal hemoglobin has a higher oxygen affinity than adult hemoglobin
- adult hemoglobin has 6 residues at the 2,3BPG binding site fetal hemoglobin has 4
- decreased affinity for 2,3BPG translates into higher oxygen affinity for fetal hemoglobin
- lower affinity for the allosteric inhibitor bestows higher affinity for oxygen
BPG: HIGH ALTITUDE ADAPTATION
- less oxygen at high altitudes
- adaption to high altitude can rapidly occur through increased production of 2,3BPG
- increased 2,3 BPG decreases hemoglobin Oxygen affinity to ensure efficient oxygen delivery to
periphery
- activity at extreme altitudes usually requires artificial oxygen

THE BOHR EFFECT

- Bohr effect describes the pH dependence of hemoglobin’s affinity of O2
- Decreased pH hemoglobin has lower affinity of O2
- Active tissues have lower pH due to:
o Increased muscle activity increases production of CO2
o CO2 eventually decreases pH
o Extreme exercise, muscles produce lactic acid to further decrease pH
- Bohr effect coordinates increased release of oxygen to active tissues
COORDINATION OF O2 DELIVERY AND CO2 REMOVAL
- Two primary challenges to cellular respiration and metabolism:
o Delivering sufficient O2 to the tissues
o Removing CO2 from the periphery
- consider that both the oxygen requirement and carbon dioxide production increase with increased
muscle activity
- body adapts effective strategies to coordinate these needs
 COORDINATION OF O2 DELIVERY AND CO2 REMOVAL: MECHANISM #1
- CO2 is taken up into red blood cells and converted to bicarbonate and a proton by enzyme
carbonic anhydrase
- Through this reaction:
o CO2 converted into a soluble form for transport to the lungs
o Decreased pH decreases hemoglobin’s O2 affinity to promote O2 release to active tissue
- The more active the tissues, the greater the production of CO2, the greater the production of CO2
the greater the releasee of O2
COORDINATION OF O2 DELIVERY AND CO2 REMOVAL: MECHANISM #2
- CO2 can form a covalent carbamate linkage to the N terminus of each chain of hemoglobin chain
to form carbaminohemoglobin
- Reaction has three important outcomes:
o Converts CO2 into a more soluble form to assist in its transport to the lungs
o Carbamino hemoglobin has a lower O2 affinity than hemoglobin to promote O2 release
o Release proton promotes O2 release through the Bohr effect
SICKLE CELL ANEMIA US A MOLECULAR DISEASE OF HEMOGLOBIN
- From a single amino acid change (Glu6Val)
- Formation of fibers from the deoxy forms of Hemoglobin
- Fibers tend to from in the capillaries which blocks blood flow to the extremities of the body
SICKLE CELL ANEMIA: MALARIA
- Sickle Cell Anemia primarily affects African Americans and Africans; selected for regions where
malaria imposes a selection pressure
- Heterozygous for Sickle Cell Anemia have resistance to malaria
- Malaria infects red blood cells
- Infection decreases pH in red blood cells
- Decreased pH causes release of oxygen from hemoglobin
- Individuals with Sickle Cell Anemia, deoxy Hemoglobin forms fibers deforming the red blood
cells
- Deformed red blood cells (contain malaria) are selectively destroyed by the spleen
OTHER OXYGEN TRANSPORT PROTEINS: HEMOCYANIN
- Invertebrates use hemocyanin rather than hemoglobin to carry oxygen
- Oxygen transporter, hemocyanin is distinct from hemoglobin in that:
o Hemocyanin uses copper rather than iron (blue blood rather than red)
o Two copper atoms bind a single oxygen molecule
o No heme ring groups, copper atom is coordinated through histidine specialized oxygen
transport cells

Module 6: Enzymes
ENZYME: INTRODUCTION
- life depends on the ability to efficient and selectively catalyze chemical reactions
- most bio molecules are very stable with rates of uncatalyzed transformations that are too slow to
permit life
- enzymes provide mechanism for acceleration, regulation, coordination of these reactions
- enzymes are their catalytic power and specificity
- side reactions leading to useless or dangerous molecules must be avoided
- Some enzymes are information sensors as well as catalyst
ENZYME: VITALISM
- biochemical reactions were believed to be inseparable from life
- Vitalism is the belief that living things are fundamentally different from non-living things
- vitalism had some famous supporters (louis Pasteur)
 - Eduard Buchner demonstrated that dead yeast still converts sugars into alcohol, indicating the
reactions of life were separate from life
- factor in yeast catalyzing the reaction, "enzyme" comes from Greek word yeast
- Eduard Buchner won noble prize for this work, ten years before he was killed in WWI
ENZYME: COENZYMES AND COFACTORS
- Proteins are well suited to form a variety of complex 3D structures that enable binding of a
variety of substances
- some enzymes, the protein component alone is fully active
- other enzymes require cofactors or coenzymes for activity
- coenzyme or cofactor that is tightly associated with the enzyme is called prosthetic group
- Different enzymes that use the same coenzyme usually perform similar types of reactions
CATALYST: GENERAL
- lower amount of energy required for a reaction to proceed
- sped up attainment of equilibrium but do not change equilibrium
- unchanged by the reaction, recycled to participate in another reaction
- enzymes offer incredible catalytic power in the rate enhancements they provide
CATALYST: ENZYMES VS CHEMICAL CATALYST
- Speed: enzymes are often much faster than chemical catalysts, some approaching catalytic
perfection
- conditions: many chemical catalysts that require extremes of temperature, pressure and pH while
enzymes function under physiological conditions
- specificity: enzymes have higher degree of specificity than most chemical catalysts, includes
specificity for what they act upon, and they produce
- regulation: unlike chemical catalysts, many enzymes are responsiveness to the dynamic needs of
the cell and organism
ENZYME: CIRCE EFFECT
- enzyme rates of catalysis can approach the physical limit rates of diffusion of molecules in
solution
- some enzymes have rate determining steps that are roughly as fast as the binding of substrates to
enzymes
- some enzymes catalyze reactions faster than predicted by diffusion control limits
- Circe effect → Greek mythology who was renowned for her ability to draw her enemies to her,
then transform them into animals
ENZYME: EQUILIBRIUM AND ES COMPLEX
- enzymes catalyze the interconversions of substrate and product
- substrate: molecule acted upon by the enzyme
- Products: molecule produced by the enzyme
- active site: the portion of enzyme responsible for binding the substrate to formation of an enzyme
substrate (Es) complex
ENZYME: ACTIVE SITE
- 1. active site is a 3D cleft formed from different parts of the polypeptide chain
- 2. active site represents just a small part of the enzyme
- 3. active sites are unique microenvironment
- 4. substrates are bound to enzymes by multiple weak interactions
- 5. specificity of substrate binding depends on the precisely defined arrangement of the atoms in
the active site.
- enzymes and their active sites can be quite flexible. Substrate binding can cause "induced fit" or
"conformation selection"
- enzyme specificity - lock and key or hand in glove
ENZYME: FREE ENERGY (RATES AND EQUILIBRIUM)
 - a reaction is spontaneous only if G is negative. Spontaneous means reaction will proceed without
input of energy and reaction releases energy
- a reaction cannot take place spontaneously if G is positive.
- a system at equilibrium, there is no net charge in the concentration of the products and reactants,
and G is zero.
- G of a reaction depends only on the free energy of the product minus the free energy of the
reactants.
- G provides no information about the rate of reaction.
- activation energy G between sand P determines the rate at which equilibrium is reached
- enzymes provide an alternate, lower energy pathway between the substrate and product lowering
G
- relationship between rate of a reaction and activation energy is inverse and exponential
- difference in free energy between sand determines the equilibrium of the reaction
- enzymes do not influence the difference in free energy between sand P and there for did not
influence the equilibrium

ENZYME: RATE ENHANCEMENT AND EQUILIBRIUM

- enzymes provide a lower energy pathway between the substrate and product, decreasing the
activation energy of the transition state and increasing rate of reaction
- enzymes do not affect the difference in free energy between the substrate and product and
therefor do not influence the equilibrium of a reaction
ENZYME: MODES OF ENZYME CATALYST
- Catalytic capabilities of enzymes result from both chemical and binding effects
- Binding effects substrate binding, transition state stabilization
- chemical effects - acid/base catalysis, covalent catalysis
BINDING EFFECTS: REACTION SPECIFICITY AND CATALYST
- binding of substrate in active site provides specificity and catalytic power
- Catalytic mechanisms limited to binding properties can still increase reaction rates by over 10000
fold
- Binding effects – 1→ substrate binding 2→transition state stabilization
- E + S > ES > ETS > E+P
- conceptual overlap between substrate binding and transition state and stabilization
BINDING EFFECTS: SUBSTRATE BINDING
- substrate binding promotes reaction by:
o reducing entropy (decreased freedom of motion of two molecules in solution)
o alignment of reactive functional groups of the enzyme with the substrate
o desolation of substrate (removal of water molecule) to expose reactive groups
o distortion of substrates
o Induced fit of the enzyme in response to substrate binding
BINDING EFFECTS: TRANSITION STATE (TS) STABILIZATION
- increased interaction of the enzyme and substrate occurs in the transition state
- essence of catalysis is stabilization of transition state
- enzyme disports the substrate, forcing it toward transition state
- active site complementary to transition state in shape and chemical character
- enzymes may bind their transition states 10 to 10 times more tightly than their substrate
- active site must be similar enough to substrate to ensure specificity, different enough to promote
change
TRANSITION STATE ANALOGS: COMPETITIVE INHIBITORS
- transition state analogs (TSAs) are stable compounds that resemble unstable transition states
- potential therapeutic applications as competitive inhibitors
 - molecules that bind to active site of an enzyme, resemble substrate molecule
- can bind to active site with high affinity, prevents substrate binding
ENZYMATIC CATALYSIS: CHEMICAL EFFECTS
- after substrate binding, enzyme can act upon the substrate to promote formation of the product
- active site often contains chemically reactive side chains
- includes polar, ionizable sidechains (triprotic) such as Asp, Glu, His, Cys, Tyr, lys, Arg, ser
- 2 commonly observed chemical catalysis:
o acid/base catalysis
o covalent catalysis
CHEMICAL MODES OF ENZYMATIC CATALYSIS: ACID BASE CATALYSIS
- reaction acceleration is achieved by catalytic transfer of a proton
- side chains of some amino acids can act as either base (proton acceptors or acids (proton donors)
- histidine with a pKa near physiological pH, often involved in acid/base catalysis
- pka of a functional group is influenced by the chemical microenvironment
- functional group of amino acids can have different pKas within active site, therefore more
suitable for acid base catalysis
CHEMICAL MODES OF ENZYMATIC CATALYSIS: COVALENT CATALYSIS
- substrate is covalently bound to the enzyme to form reactive intermediate
- covalent catalysis often involves two steps, first forms a covalent linkage to the enzyme, second
regenerate the free enzyme
COVALENT CATALYSIS: SUCROSE PHOSPHORYLASE
- step one: glucosyl residue is transferred to enzyme glucose fructose + Enz → s glucosyl- Enz +
Fructose
- Step two: glucose is transferred to phosphate glucosyl - Enz + Pi → glucose 1-phosphate + Enz
ENZYME KINETICS: GENERAL
- Kinetics: study of velocity of reactions substrate → product
∆[𝑃]
- velocity of a reaction is qualified as the change in concentration of product over time 𝑣 = ∆𝑡
- enzyme kinetics measured in units of concentration overtime
ENZYMES KINETICS: VARIABLES THAT ENZYME VELOCITY
- enzymes are proteins, any variable that influences protein structure may influence enzyme
activity
- activity of enzyme is temperature and pH sensitive
- enzymes can have different optimum temperatures and pHs
- enzyme velocities are also influenced by enzyme and substrate concentration
- Kinetics - interested in relationship between velocity and substrate concentration
KINETICS: INITIAL VELOCITY
- velocity - change in product concentration over time, necessary to measure product formation
before equilibrium is reached
- Vi is velocity at the beginning of an enzyme catalyzed reaction, before product accumulation
- K1 and K2 represent rapid, noncovalent interactions between enzyme and substrate
- K2 rate constant of formation of product from ES
𝐾1→ 𝐾2−→
- 𝐸 + 𝑆 <−𝐾−2 𝐸𝑆 <−𝐾−2 𝐸 + 𝑃 Vo=[ES]K2
MICHAELIS-MENTON KINETICS: STEADY STATE ASSUMPTION
- Worked from the assumption that the rate of formation of the ES complex was equal to the rate of
its breakdown (steady state assumption)
𝐾1→ 𝐾2−→
- 𝐸+𝑆 𝐸𝑆 𝐸+𝑃
<−𝐾−2 <−𝐾−2
- Mathematically steady state assumption states that: [E][S]K1=[ES]K-1 = [ES] K2
o Rate of formation of the ES complex is [E] [S] K1
o Rate of breakdown of the ES complex is [ES]K-1 + [ES]K2
MICHAELIS-MENTON: EQUATION AND PLOT
𝑉𝑚𝑎𝑥 [𝑆]
- describe the relationship between substrate concentration and initial velocity 𝑉𝑜 = 𝐾𝑚+[𝑆]
- km → concentration of substrate required to reach ½ Vmax
- Vmax → maximum velocity of the enzyme
MICHAELIS-MENTON: KM
- concentration of substrate required for enzyme to function at half maximal velocity
- many enzymes, Km provides an accurate approx. of in vivo substrate concentration
- most enzymes usually functioning at about half their maximum velocity
- [S]<km, enzymes are highly sensitive to changes in substrate concentration but have little activity
- [S] >km, enzymes have high activity but are insensitive to changes in substrate concentration
- [S] = km, enzyme has significant activity and is responsive to changes in substrate concentration
KINEITCS: LINEWEAVER BURK PLOT
- describe relationship between [S] and Vo
- double reciprocal plot of 1/Vo vs 1/[S]
- precise method of analysis of kinetic data
1 𝐾𝑚 1
- used to determine Vmax and km 𝑉𝑜 = 𝑉𝑚𝑎𝑥 [𝑆] + 𝑉𝑚𝑎𝑥
KINETICS: ENZYME TURN OVER NUMBER
- called Kcat
- equals the number of molecules of substrate converted to product per unit time under saturating
conditions
- calculated by Vmax/[E+]
REVERSIBLE ENZYME INHIBITION: GENERAL
- inhibitor is a compound that binds to an enzyme to interfere with its activity
- inhibitors can prevent formation of ES or the breakdown to E and P
- reversible inhibitor bind to the enzyme by non-covalent interactions
- two classes of reversible enzyme inhibitors with different mechanisms and kinetic consequences
competitive → uncompetitive → non-competitive
REVERSIBLE ENZYME INHIBITION: COMPETITIVE
- competitive inhibitors resemble the substrate and compete with the substrate for binding active
site
- antibiotic sulfanilamide is a competitive inhibitor of abacterial enzyme that has PABA as a
substrate
- competitive inhibitors bind only the free enzyme
- effect of competitive inhibitors can be overcome with an excess of substrate
- Vmax is the same but apparent km is increased
REVERSIBLE ENZYME INHIBITION: UNCOMPETITIVE
- Bind only to the ES complex
- Vmax is decreased by conversion of ES to ESI, cannot form product
- Reduce [ES]
- E binds to S to replenish ES this apparent increase in affinity of the E for S causes a decrease in
Km
- Herbicide “round up” is an uncompetitive inhibitor of a plant enzyme involved in amino acid
metabolism
REVERSIBLE ENZYME INHIBITION: NON-COMPETITIVE
- non-competitive inhibitors bind to E and Es
- Vmax is decreased with no change in km
- non-competitive inhibitors don't influence s binding, no change in km
- reduces the number of active enzyme molecules
- antibiotics doxycycline is a non-competitive inhibitor of bacterial enzyme
SERINE PROTEASES: GENERAL PROPERTIES
- serves as a digestive enzyme, including trypsin, chymotrypsin, and elastase, that cleave peptide
bonds in protein substrates
- members of this family share similar sequences and active site residues
- synthesized and stored in the pancreas as inactive zymogens to prevent damage to cellular
proteins
- catalytic mechanism contains elements of both covalent and acid base catalysis
SERINE PROTEASES: SUBSTRATE SPECIFICITIES
- has unique specificities that reflect unique substrate binding pockets
- thrombin cleaves Arg-Gly bonds
- Trypsin cleaves by Lys and Arg
- Chymotrypsin cleaves by Phe, Tyr or Met
- Elastase cleaves by Gly and Ala
- Papain cuts all peptide bonds
SERINE PROTEASE: CATALYTIC TRIAD
- Serine proteases have a conserved catalytic mechanism based on a catalytic triad of residues
(Asp, Hid, Ser)
- His acts to accept and donate a proton at each of the two stages of the reaction mechanism (acid
base catalysis)
- Asp stabilizes the positively charged His to facilitate serin ionization
- Ser attacks the carbonyl group of the peptide bond to be cleaved (covalent catalysis)
CHYMOTRYPSIN MECHANISM: OVERVIEW
- Phase 1:
o Step 1: acid/base, histidine acts as a base to extract proton from hydroxyl of Ser.
Activates the oxygen of the hydroxyl group
o Step 2: covalent, formation of covalent linkage from the hydroxyl group of the Ser to the
carbonyl carbon of the peptide bond to be cleaved in the substrate
o Step 3: acid/base, histidine acts as an acid to donate a proton to the amine group of
peptide bond to be cleaved, this cuts the substrate peptide into two pieces
- Phase 2:
o Step 1: acid/base, histidine acts as a base to extract a proton form a water molecule,
activating the oxygen of this molecule
o Step 2: covalent, activated water molecule attacks the point of covalent linkage between
enzyme and substrates
o Step 3: acid/base, histidine acts as an acid to donate a proton to reform the hydroxyl
group of Ser

REGULATION OF ENZYME ACTIVITY: OVERVIEW

- Activity of an enzyme can be regulated by controlling the amount of the enzyme (long term)
- By adjusting the activity of a constant quantity of the enzyme (short term)
- Regulation of enzyme availability: location, rates of synthesis and degradation
- Regulation of enzyme activity:
o Covalent modification: phosphorylation, methylation, glyosylation, etc
o Noncovalent modification (allosteric): allosteric regulation
REGULATION OF ENZYME ACTIVITY: POINTS OF REGULATION
- Enzymatic pathways often controlled through negative feedback inhibition by the final product of
the pathways
- Final product often inhibits the enzyme catalyzing the first unique and committed step
- Regulation at this step conserves material and energy and prevents accumulation of intermediates
- F is the end product is needed in limited amounts and cannot be stored
 - A is values and showed be conserved unless F is needed
- B,C,D and E, have no biological roles other except as intermediates in the production of F
REGULATION OF ENZYME ACTIVITY: POINTS OF REGULATION
- Negative feedback in branched pathways
o Often occurs by the final product of each branched acting to inhibit the enzyme
catalyzing the first unique and committed step of the branch
- Regulation when two pathways cooperate to form a single product
- Final product can inhibit the first unique step of each branch
- Molecules preceding the merger can inhibit the first step of their branch as well as activating the
first step of the opposing branch
ALLOSTERIC ENZYMES: GENERAL PRPOERTIES
- Information sensors to coordinate cellular metabolism
- Regulated by interaction with metabolic intermediates
- Regulated by allosteric modulators that bind non-covalently at sites other than the active sites
- Examples of quaternary structures
- Catalyze branch point reactions
- Often slow, representing the rate limiting step of pathway
- DO NOT OBEY MICHAELIS-MENTEN KINETICS
o Instead they have sigmoidal curves
- Activities of allosteric regulator enzymes are changed by inhibitors and activators (modulators)
- Allosteric modulators bind non-covalently to the enzymes that they regulate
- Regulatory enzymes often possess quaternary structure
- Rapid transition between the active (R) and inactive (T) conformations
- Substrates and activators many bind only to the R state while inhibitors many bind only to the T
state
- Binding of the substrate disrupt the R to T equilibrium in favor of R
o Basis to the co-operative activation of allosteric enzymes
ALLOSTERIC ENZYMES: PHISIOLOGICAL SIGNIFICANCE OF COOPERTIVITY
- Allosteric enzymes transition from less active state to a more active state within narrow range of a
substrate concentration
- Activity of allosteric enzymes is more sensitive to changes in substrate concentration near the Km
than are Michaelis-Menten enzymes of the same Vmax
- Sensitivity is called threshold effect: below a certain substrate concentration there is little enzyme
activity
o After threshold is reached the enzyme activity increases rapidly
ALLOSTERIC ENZYMES: PHOSPHOFRUCTOKINASE 1
- Catalyzes an early step of glycolysis
- Phosphoenolpyruvate (PEP), an intermediate near the end of the pathway is an allosteric inhibitor
of Phosphofructokinase 1
- ADP is an allosteric activator of phosphofructokinase1
- Ratio [PEP]/[ADP] is high, phosphofructokinase 1 is inhibited
o Ratio of [PEP]/[ADP] is low, phosphofructokinase 1 is activated and glycolysis produces
more ATP and ADP
- Concentrations of PEP and ADP act allosterically through phosphofructokinase 1 regulate the
activity of the entire pathway
ALLOSTERIC ENZYMES: ACTIVATION OF PHOSPHOFRUCTIKINASE 1 BY ADP
- Activity of phosphofructokinase 1 is responsive to concentration of the substrate as well as the
allosteric activators and inhibitors
- At constant levels of substrate the activity of the enzyme can be modulated through changes in
levels of the allosteric modulators
ENZYME REGULATION BY COVALENT MODIFICATION: GENERAL
 -Enzymes are regulated through the covalent linkage of a modifying group to changes some aspect
of the proteins behavior
- Number of different types of covalent modification have been characterized
- Most common post translation covalent modification is through phosphorylation
- Modifications are usually reversible with one enzyme catalyzing the addition of the group and
another enzyme catalyzing its removal
- Kinases add phosphoryl groups, phosphatases remove them
ENZYME REGULATION BY COVALENT MODIFICATION: GLYCOGEN METABOLISM
- Production and utilization of glycogen is controlled by two enzymes:
o Glycogen synthase (anabolic) which catalyzes production of glycogen from fructose
o Glycogen phosphorylase (catabolic) which catalyzes the breakdown of glycogen into
glucose
- Glycogen to glucose: in response to hormones that are released when you are hungry (glucagon)
or scared (epinephrine) both enzymes are phosphorylated
o phosphorylation activates the catabolic enzyme and inactivates the anabolic enzyme
o situation favors the breakdown of glycogen into glucose
- glucose to glycogen: in response to hormones released in the fed state (insulin) both enzymes are
both unphosphorylated
o when unphosphorylated the anabolic enzyme is active, and the catabolic enzyme is
inactive
o situation favors the storage of glucose within glycogen

Module 7: Carbohydrates
CARBOHYDRATES: PHYSIOLOGICAL FUNCTIONS
- Physiological roles of carbohydrates
o Energy- burn sugars to produce energy to do the processes
o Energy storage- glycogen (storage of glucose)
o Structural roles- cellulose, fibrous material that exist in plants (no skeleton)
o Cellular interactions- in between all cells complex network, large and elaborate
carbohydrate structure (properties associated with that), present in joints, serve as a
lubricant
o Cellular identification- outside of cell looks the same but how can you tell where it is
from, health status
o Information transfer (DNA & RNA)- within every nucleotide you have sugar
o Signalling- post translation
MONOSACCHARIDE FAMILIES: ALDOSES AND KETOSES
- Every carbon you have, there is a water molecule associated with it
- Carbohydrates have empirical formulas of (CH2O)n, where n is greater than or equal to 3
- Two major classes of carbohydrates:
o Aldoses- aldehyde groups
o Ketoses- ketone group
- Everyone will have a carbonyl
o Where it is positioned tells you if it is aldose or ketose
o If it is at the end of the structure, it is a aldose sugar
o If it is in the middle of the structures, it is a ketose sugar
MONOSACCHARIDES: CHIRAL CARBON
- Monosaccharides have multiple asymmetric carbons
- Designation of a sugar as either D or L is based on the configuration of the chiral carbon most
distant from the carbonyl group
- Designation of the D or L is relative to the reference molecule D-glyceraldehyde
- Molecules with n chiral centers will have 2n stereoisomers
 - Number of categories of isomers within carbohydrates
- Epimers: two molecules that differ in configuration by 1 and only chiral carbon
- Anomers: see sugars undergo cyclization events which create new chiral carbons
o Differ in configuration about the carbon that becomes chiral consequences of cyclization
MONOSACCHARIDES: EPIMERS
- Epimers are sugars that differ at a single chiral center
o Where the molecule switches
MONOSACCHARIDES: CYCLIC STRUCTURES
- Longer carbohydrates (5 carbons and up) tend to be cyclized
- Formation of these cyclized structures is the results of a reaction of an internal alcohol (hydroxyl
group) with either:
o Aldehyde (aldoses) to form a hemiacetal
▪ Aldehyde + alcohol = hemiacetal
o Ketone (ketoses) to form a hemiketal
▪ Ketone + alcohol = hemiketal
CYCLIC MONOSACCHARIDES: PYRAN AND FURAN RING STRUCTURES
- Cyclized carbohydrates can be found in two ring forms
o Six ring membered sugar ring is called “pyranose”
o Five ring membered sugar ring is called “furanose”
CYCLIZATION OF MONOSACCHARIDES: GLUCOSE TO GLUCOPYRANOSE
- Cyclization of glucose involves the C5 hydroxyl and C1 aldehyde
- Cyclization renders C1 chiral, produces two stereoisomers, alpha and beta
- Carbon that becomes chiral as a result of cyclization is the anomeric carbon
- Alpha and beta forms are anomers of each other
ANOMERIC CARBONS: ALPHA AND BETA CONFIGURATIONS
- Cyclization produces a new chiral carbon at the anomeric carbon resulting in two new
stereoisomers
- Alpha hydroxyl of anomeric carbon is below the plane of the sugar
- Beta hydroxyl of anomeric carbon is above the plane of the sugar
ANOMERIC CARBONS: MUTAROTATIONS
- Alpha and beta configurations interconvert by a process called mutarotation
- Mutarotation occurs through a linear intermediate
- Mutarotation represents a change in configuration
- glucose tends to be 2/3 beta glucopyranose, 1/3 beta glucopyranose and less than 1% linear
CYCLIZATION OF MONOSACCHARIDES: D-FRUCTOSE TO FRUCTOFURANOSE
- cyclization of fructose involves the C5 hydroxyl and the C2 Ketone
- cyclization renders C2 chiral (anomeric carbon), producing alpha and beta stereoisomers
- Cyclization always involves a carbonyl carbon
RING FORMS: FRUCTOFURANOSE AND FRUCTOPYRANOSE
- Fructose exists in both pyran and furan rings
- Beta-fructopyranose (found in hone), is extremely sweet
- Heating the beta-fructopyranse converts it to the beta-fructofuranose (less sweet)
- Carbon syrup, high concentration of beta-fructopyranose, sweetens cold drinks but not hot drinks
SUGAR DERIVATIVES: GENERAL
- Carbohydrates derivatives break from the empirical formula of (CH2O)n
- Derivatives can include nitrogen, phosphate and sulfur groups
- Modifications usually indicate specialized functions
SUGAR DERIVATIVES: MUSTARD BOMBS
- Plants contain glucosinolate and the enzyme myrosinase
- Myrosinase acts on glucosinolate to produce glucose and isothiocyanate
- Myrosinase and glucosinolate are stored separately, come together upon tissue damage
 - Bitter taste functions to discourage herbivores from eating the plant
MONOSACCHARIDES: REDUCING AGES
- Linear forms of monosaccharides can be oxidized by mild oxidizing agents (iron and copper)
- Carbonyl group is oxidized to a carboxyl group
- Quantifications of sugars presents in blood and urine
- Defines the end of the sugar with the carbonyl carbon as being the reducing age
DISACCHARIDES: NOMENCLATURE
- Glycosidic bond: primary structural linkage in all polymers of monosaccharides
o O-glycosidic bonds occur through oxygen
o N-glycosidic bonds occur through nitrogen
- Nomenclature of a disaccharide specificity:
o Monosaccharides involved in the disaccharide
o Their ring types (puran, furan)
o Configurations (alpha or beta)
o Linkages (C1-C4, etc)
- End of chain with free anomeric carbon called reducing end of polymer
DISACCHARIDES: GENERAL
- Monosaccharides have multiple hydroxyl groups means that there are many different glycosidic
linkages are possible
- Three monosaccharides (glucose, galactose and mannose) can generate 12,000 different structures
- Higher order carbohydrate structures are generated through the action of glycosyltransferase
- Glycosyltransferases use monosaccharides that are activated through linkage with UDP
DISACCHARIDES: NOMENCLATURE
- Specify the configuration (alpha or beta) at the anomeric carbon of each monosaccharides
- Specify the ring form (furan or pyran) of each monosaccharide
- non-reducing sugar has the suffix “osy”
- reducing sugar has the suffix “ose”
- indicated in the parentheses the two carbon atoms joined by the glycosidic bond with an arrow
DISSHARIDES: LACTOSE
- Lactose intolerant people have insufficient levels of the enzyme (lactase) that catalyzes the
hydrolysis of lactose into glucose and galactose
- Consumption of lactose by these individuals can lead to bloating, cramps, flatulence, diarrhea and
nausea
- Mammals cease to produce lactase after weaning but human populations have developed lactase
persistence
POLYSACCHARIDES: GENERAL
- Have diverse structures and functions
- Biological functions of polysaccharides include energy storage, structural roles, cushioning and
lubrication, etc
- Homopolysaccharides: polymers containing a single type of monosaccharides
- Heteropolysaccharides: polymers containing more than one type of monosaccharide
- Both hetero and homopolysaccharides can be either unbranched or branched
ENERGY STORAGE POLYSACCHARIDES: STARCH AND GLYCOGEN
- Glucose is stored intracellularly in polymeric forms on both plants (starch) and animals
(glycogen)
- Starch and glycogen are both heavily hydrated structures
- Starch:
o Mixture of two molecules, amylose (unbranched) and amylopectin (branched)
o Found in plants and fungi
- Glycogen:
o Stored in liver and skeletal muscle of animals
ENERGY STORAGE POLYSACCHARIDES: STARCH (AMYLOSE AND AMYLOPECTIN)
- Amylose: linear polymer of glucose residues through alpha (1-4) bonds
- Amylopectin: alpha (1-4) linked glucose residues with alpha (1-6) branch points every 24-30
residues
- Alpha (1-4) cut by amylase, alpha (1-6) branch points debranching enzyme
- Amylose and amylopectin each have a single reducing end
- Amylose has a single non-reducing end
- Amylopectin has multiple non-reducing ends
ENERGY STORAGE POLYSACCHARIDES: GLYCOGEN
- Presents in all cells, most prevalent in skeletal muscle and liver
- Identical to amylopectin consists of alpha (1-4) linked glucose residues with alpha (1-6) branch
points but with a higher frequency of branch points (every 10 residues)
- Mobilized in times of need by glycogen phosphorylase which sequential cleaves glucose residues
from non-reducing ends
- Greater frequency of branching allows for rapid mobilization
STRUCTURAL POLYSACCHARIDES: CELLULOSE
- Primary component of plant cell walls (fiber)
o Accounts for over half of the carbon in the biosphere
- Is a linear, homopolysaccharides of glucose residues
- Has a linear arrangement of beta (1-4) glycosidic residues
o Bonds cannot be cut by amylase
STRUCTURAL POLYSACCHARIDES: CHITIN
- Principal component of hard exoskeletons (insects, lobster, etc)
- Linear homopolysaccharides of N-acetylglucosamine residues
- Chemical difference between cellulose and chitin is the replacement of hydroxyl group at C2 with
acetylated amino group
POLYSACCHARIDES: ALPHA AND BETA LINKAGES
- Beta (1-4) linkage of cellulose and chitin allow formation of long straight chains
- Fibrils are formed by parallel chains that are linked through hydrogen bonds
- Fibers generate a rigid supportive structure of high tensile strength
- Alpha (1-4) linkage of starch and glycogen form a hollow, helical structure
- Hollow helix provides a compact, accessible storage structure of glucose
GLYCOLIPIDS: BLOOD GROUP ANTIAGENTS
- Sugars are also covalently linked to lipid molecules to form glycolipids
- Central function of glycolipids is in the blood group antigens
- Different patterns of sugars presented on the surface of cells by the glycolipids help the body to
discriminate self from non self
- Differences in the blood group antigens critical for blood transfusions
PROTEINS-ASSOCIATED CARBOHYDRATES: GENERAL
- Glycoproteins:
o Proteins with covalently attached sugars
o Protein constituent is the largest component by weight
o Serve a variety of biological roles
- Proteoglycans
o Protein component is linked to a particular type of carbohydrate called a
glycosaminoglycan
o Carbohydrate consistent is the largest component by weight
o Often serve structural and lubricating functions
GLYCOPROTEINS: GENERAL
- Proteins undergo post- translational glycosylation (adding sugar groups)
- Half biological proteins are modified with sugar groups
 o Modifications regulate the structure and function of protein
o Modifications involve complex specific patterns of sugar attachment
- Sugars are attached either to:
o Amide nitrogen of the aide chain of an asparagine (N-linked)
o Hydroxyl oxygen of the side chain of serine or threonine (O-linked)
GLYCOPROTIENS: ERYTHROPOIETIN (EPO)
- Stimulates red blood cell production
- Three N-linked and O-linked glycosylation
- High effective in linked treatment of anemia (red blood cell deficiency)
- Exploited by endurance athletes to increase oxygen carrying capacity
- Patterns of glycosylation associated with the synthetic version of EPO differ from those of the
endogenous protein
PROTEOGLYCANSL: GENERAL
- Extra cellular space in tissues contains a gel like material
o The extra cellular matrix (ground substance)
▪ Ground substance holds cells together
▪ Provides porous pathways for diffusion of nutrients/ waster products
▪ Serves as a cushioning function
- Extra cellular matrix is a meshwork of fibrous proteins and heteropolysaccharides called
glycosaminoglycans

Module 8: Lipids
LIPIDS: GENERAL
- Functional characteristics
o Lipids play a number of diverse biological function including:
▪ energy storage (fat in animals, oil in plants)
▪ structural component of membranes
▪ active roles
• signalling (messengers inside cells, between cells and between tissues)
• enzyme co-factors & vitamins
- everything that is insoluble in water
- nonpolar, hydrophobic molecules
- not going to form polymers
- DNA link together all nucleotides
- Membranes have highly specialized structures
- Types of molecules that are making them up

FATTY ACIDS ARE HYDROCARBON DERIVATIVES

- Basic structure of fatty acid
- All have a carboxyl group
- Hydrocarbon tail
- amphipathic molecules
- Polar group
- Fatty acids (general)
o Hydrocarbon with carboxylic head
o Differ in length and degree of saturation
o Usually an even number of C’s (12-24)
- Double bonds within fatty acids:
o Saturated (no double bond)
o Unsaturated (1 double bond)
o Polyunsaturated (multiple double bonds)
 o Double bonds usually cis configuration
o Double bonds usually separated by methylene group
FATTY ACID NOMENCLATURE
- Nomenclature of fatty acids address the potential points of variability
o Length
o Presence or. Absence of double bonds
o Location of double bonds
( # CARBONS: #DOUBLE BONDS, ∆DOUBLE BOND POSITION )
- Position of double bond indicated by ∆n , where n is the lowest numbered carbon involved in
double bond
- Carbon of the carboxyl group is the carbon 1
FATTY ACID STUCTURE
- Hydrocarbon tails of fatty acids associate through hydrophobic and Van Der Waals interactions
- Long chains have stronger associations than short chains
- Saturated chains have stringer associates than unsaturated
ASSOCIATION OF FATTY ACIDS
- Melting temperature of a fatty acid mixture reflects the length and degree of saturation of the
hydrocarbon tails
- Double bonds have greater influence than the length of the tail on the fatty acid association
FATS AND OILS AS ENERGY STORAGE MOLECULES
- Lipids represent critical energy storage molecules for plants and animals
- Lipids occupy most of the intracellular space in adipocytes
o Energy storage is similar in animals
- Storage of fat under the skin also provides an insulating function or cold weather animals
- Fatty acids required for energy are stored as triacylglycerol
TRIACLYGLYCEROLS ARE FATTY ACID ESTER OF GLYCEROL
- Triacylglycerols: storage lipids in animals and plants
- Three fatty acids linked to glycerol through ester linkages
- Triacylglycerols have the same three fatty acids at each position, complex triacylglycerols have
different fatty acids
- Ester linkages removes the polar carboxyl group to make a more hydrophobic molecule
- Getting rid of the negative charge that existed on the carboxyl group
o Makes molecule less polar
A PERSPECTIVE ON ENERGY STORAGE MOLECULES
- Different energy storage molecules serve different biological roles
- Fats represent the key molecules for long term energy storage
- A gram per gram basis
o Six times as much energy in fats than in carbohydrates
- Low oxidation state: less oxygenated fuels burn more efficiently; triacylglycerols have lower
oxygenation state than carbohydrates
- Low hydration state: lipids are hydrophobic with limited interaction with water providing a more
compact, dehydrated energy storage from
MAKING SOAP FROM FAT
- Saponification: Fat with a strong base breaks the ester linkages to release free fatty acids
- Amphipathic properties of free fatty acids make them effective in solubilization of hydrophobic
substances
- Fatty acids function as detergents and soaps through formation of micelles that capture
hydrophobic molecules
BETTER LIVING THROUGH SCIENCE
-
WAXES SERVE AS ENERGY RESERVES AND WATER REPELLENTS
 - Waxes are non-polar esters of long chain fatty acids and long chain monohydroxylic alcohols
- Waxes are water insoluble and have high melting temps
- Widely distributed in nature as protective waterproof coatings on leaves, fruit, animal skin and
feathers
STRUCTURAL LIPIDS IN MEMBRANES
- Membrane bilayers define cells and regulate the composition of the intracellular environment
- Formation of membranes is a spontaneous consequence of the properties of the molecules that
compose them
GLYCEROPHOSPHOLIPIDS
- Lipid components of membranes tend to have similar overall shapes and properties
o Two hydrophobic tails and hydrophobic head group
- Membrane lipids can be classified based on their backbone (glycerol vs sphingosine) or by their
polar head groups (phosphor vs glycol)
GALACTOLIPIDS AND SULFOLIPIDS
- Most abundant lipids in membranes
- Glycerol backbone with phosphate at the C3 position (glycerol 3-phosphate)
- Glyercol-3-phosphate + 2 fatty acids= phosphatide
SPHINGOLIPIDS
- Sphingolipids are derived from sphingosine (long chain of amino acid)
- Sphingosine is similar to monoacyl glycerol
- Single fatty acid linked by amide bond to form ceramide
- Ceramide is similar to diacylglycerol
- Glycerophospholipids have a variety of polar head groups can be attached to cermide
- Common sphingolipids include
o Sphingomyelins phosphocholine
o Cerebrosides
▪ Glycosphingolipids
▪ Single sugar unit
o Gangliosides
▪ Glycosphingolipids
▪ Multiple sugar units
GLYCOSPHINGOLIPIDS DETERMINE BLOOD TYPE
- cells are recognized as “self” vs “nonself” based on patterns of surface exposed carbohydrates
- transfusion of an incompatible blood type causes severe immunological reaction
- different blood types (A, B, O) reflect different sugar patterns as the head groups of the
sphingolipids
UNIQUE MEMBRANE LIPIDS
- Archaebacteria live under conditions of high temp, pH and ionic strength
o This makes it a challenge to maintain the membrane integrity
- Membrane lipids of these extremophiles often contain
o Ethers linkages
o Branch points within the hydrocarbon tails
o Membrane spanning hydrocarbon tails composed from a single molecule

STEROIDS
- Sterols are structural membrane lipids
- Sterols contain four fused ring steroid nucleus: 3-six carbon rings and a 5-carbon D ring
- Ring system is rigid and nearly planar
- Sterols serve as precursors for many biologically active products (testosterone)
CHOLESTEROL
 - Cholesterol serves as a number of critical biological functions such as mediating membrane
fluidity
- Serves as a precursor of steroid hormones and bile salts
LIPIDS AS SIGNALS, COFACTORS AND PIGMENTS
- The lipids we have been considering have primarily passive roles
o Energy storage and membrane structure
- Active roles for lipids include:
o Intracellular signaling molecules
o Hormones
o Enzyme cofactors
o Pigments
o vitamins
PHOSPHATIDYLINOSITOLS ACT AS INTRACELLULAR SIGNAL
- phosphatidylinositol 4,5-bisphosphate on inner membrane face
- activation of the phospholipase C in response to an extracellular signal trigger the cleavage of
head group to produce inositol 1, 4, 5 triphosphates, then increases to Ca2+
- both Ca2+ and diacylglycerol activate specific intracellular pathways and processes
EICOSNOIDS
- Eicosanoids
o Paracrine hormones (act on cells near point of production)
o Derivatives of C20 polyunsaturated fatty acids (arachidonic acid)
o Three classes of eicosanoids
- Prostaglandins
o Contraction of blood vessels
- Thromboxane
o Involved in blood clot formation
- Leukotrienes
o Smooth muscle contraction
STEROID HORMONES
- Hydrophobic oxidized sterol derivatives
- Carried through blood stream by carrier proteins
- Pass through plasma membrane to bind receptors in the nucleus
- Alter patterns of gene expression and metabolism
LIPID VITAMINS
- Four lipid vitamins : A, D, E, K
- All contains rings and long, aliphatic side chains
- All are highly hydrophobic
- Lipid vitamins differ widely in their functions
VITAMIN D
- Regulates Ca 2+ uptake and deposition
- Can be obtained from the diet or produced endogenously
- Endogenous production occurs in a series of reactions
o One of which requires UV light
- Insufficient vitamin D is associated with skeletal defects
- Excessive vitamin D can cause calcification of soft tissues

VITAMIN A
- Obtained by liver, egg yolks and milk products
- Some animals have sufficient vitamin A in their livers to present a danger to humans in they were
to consume them
- Exists in 3 forms:
 o Alcohol (retinol)
o Aldehyde
o Retinoic acid
- Retinal (aldehyde) is a light sensitive compound with a role in vision
VITAMIN E (ALPHA-TOCOPHEROL)
- Reducing reagent that scavenges oxygen free radicals
- Prevent damage to fatty acids in membranes
- Used as an addictive in cosmetics
- Deficiency causes:
o Scaly skin
o Muscular weakness
o Sterility
VITAMIN K
- Required for synthesis of blood coagulation proteins
- Warfarin was used as rat poison where it cause rodents to suffer uncontrolled bleeding
- Vitamin K analogs are now given individuals who suffer excessive blood clotting

Chapter 11
MEMBRANE FUNCTIONS
- Common to all life forms, have to be able to create an environment where you have different
composition of stuff than outside the cell, have to create barriers to encapsulate proteins, and
biomolecules
o Separate cells from the external medium to create an intracellular environment of unique
and define composition
o Allow selective transport of substrates in and out of the cell
o Provide a location for specialized pathways and processes
▪ Energy conversion of mitochondria
o Rapid changes in electric potential across the membranes of neurons as basis of the
nervous system
o Localization of receptors to facilitate response to physiological signals
o Mediate cell to cell recognition and interaction
MEMBRANE CHARACTERISTICS
- Membranes are sheet-like structures, two molecules thick, that forms closed boundaries between
compartments
- Membranes consists of mainly of lipids and proteins, with carbohydrates linked to these
molecules
- Membranes are built from amphipathic molecules
- Membranes are largely impermeable to polar molecules
- Specific membrane proteins mediate particular biological functions
- Membranes are self-assembling, non-covalent structures
- Membranes are fluid and dynamic structures
- Membranes are highly specialized in their composition and distribution (asymmetric)
LIPID BILAYER IS THE BASIC STRUCTURAL ELEMENT OF MEMBRANES
- Membrane formation is a consequence of the amphipathic nature of the membrane lipids
- Molecules self-assemble through the hydrophobic effect
- Structure formed depends on the ratio of cross-sectional areas of the polar head group and the
hydrophobic tail
o Fatty acids favour micelle formation of the micelles
o Lipids with two hydrocarbon tails tend to form bilayers (glycerophospholipids and
sphingolipids)
LIPID BILAYERS FORM MEMBRANE VESICLES
 - Exposure of hydrophobic tails at the edge of the bilayer to water is energetically unfavorable
- Flat bilayer sheets are unstable and spontaneously form membrane vesicles with an internal
volume
- vesicles are the basis of cells and organelles
MEMBRANE ARE IMPERMEABLE TO POLAR MOLECULES
- lipid bilayers membrane have a very low permeability to ions and most polar molecules
- permeability of a small molecules is correlated with their relative solubility in water
- small non-polar gases (O2 and CO2)
o small hydrophobic molecules (fat soluble hormones) pass directly through the membrane
VESICLES FOR DRUG DELIVERY
- cell membrane can represent a critical barrier for polar drugs intended for intracellular targets
- encapsulation of a drug within a liposome can facilitate transport across the membrane
- liposomes can also be used to target specific cells or organelles
MEMBRANE COMPOSITION
- membranes are primarily composed of lipids and proteins
- more active membranes have a higher ratio of protein to lipid
- composition of membrane components can be dynamic (prokaryotes)
FLUID MOSAIC MODEL OF MEMBRANE STRUCTURE
- membranes are dynamic structures due to the nature of the non-covalent interactions
- lipids and proteins freely diffuse in the plane of the membrane
- lateral movement of proteins and lipids within the membrane is very rapid
- movement across the membrane is restricted
TRANSBILAYER MOVEMENT REQUIRES CATALYSIS
- transverse is going one side to the other
- trans bilayer movement requires a polar head group to pass through hydrophobic environment
- uncatalyzed rate od lipid molecule crossing from one sheet to the other is very slow
- translocation of lipids from one side of bilayer to the other is catalyzed by the enzyme called
flippases
- lipid composition of the inner and outer sheets of the bilayer can be different, allowing for
specialization of the membrane faces
MEMBRANE FLUIDITY
- calls need to maintain an appropriate level of membrane fluidity
- membranes undergo temperature-dependent phase transitions
o below the phase transition temp, membrane is too solid
o above the phase transition temp, membrane is too fluid
o at the phase transition temp, the hydrocarbon chains are partially ordered but lateral
diffusion still possible
- cells can adjust membrane composition to maintain liquid ordered state
o bacteria vary the length and saturation of the hydrocarbon tails of membrane lipids
o animals use cholesterol to mediate membrane fluidity
SPECILIZATION OF MEMBRANE STRUCTURE AND FUNCTION
- while basic features of the bilayer are simple and consistent, mechanisms to enable specialization
o composition of membrane components
▪ lipids
▪ proteins
o distribution of membrane components
▪ static
▪ dynamic
o specialized membrane regions
▪ lipid rafts
SPECIALIZED COMPOSITIONS AND DISTRIBUTION OF LIPIDS IN MEMBRANES
- lipid composition of membranes varies across species and cell types
o highly specialized compositions and functions
- dynamic changes to composition and/or positioning to regulate biological events
SPECIALIZED MEMBRANE REGIONS (LIPID RAFTS)
- lipid rafts arise from spontaneous association of the lipid molecules whose hydrocarbon tails are
of similar length
- sphingolipids form clusters
o exclude glycerophospholipids
- longer, saturated hydrocarbons of sphingolipids form stable associations making thicker rafts and
more ordered than the rest of the membrane
- rafts are docking points in lipid-anchored proteins that contain long chain saturated fatty acid
anchors
- lipid linked proteins that associate with rafts often serve signalling functions
MEMBRANE PROTEINS
- active roles of membranes often performed by proteins
o receptors and transport
- three catagories of membrane proteins are defined based on different mechanisms of association
with the membrane
o peripheral (c and d)
o lipid-anchored (e)
o integral membrane proteins (a and b)
PERIPHERSL MEMBRANE PROTEINS
- associate with membrane through electrostatic or hydrogen-bonding interactions
- dock to either membrane lipids or integral membrane proteins
- bulk of the peripheral membrane proteins is in the cytosol or extracellular space
- changes in pH or ionic strength often releases these proteins from the membrane
LIPID-ANCHORED MEMBRANE PROTEINS
- covalently attached lipids can anchor proteins to the membrane
- these protein modifications are sometimes reversible, allowing for regulation of cellular locations
- GPI anchored proteins always at outer face
- Proteins with single chain hydrocarbons always to inner face
INTEGRAL MEMBRNE PROTEINS
- Integral membrane proteins are immersed in, and usually span, the membrane
- Protein positioning within a membrane is specific and directional
- Integral membrane proteins tend to be of three varieties
o Single pass alpha helical
o Alpha helical bundles
o Beta-barrels
DISTRIBUTION OF AMINO ACIDS IN INTEGRAL MEMBRANE PROTEINS
- Integral membrane proteins, charged residues (blue) are located mostly within the intra and
extracellular portions of the protein
- Residues with non-polar side chains (white) dominate inside the hydrophobic slab of the bilayer
- Tryptophan (red) and tyrosine (orange) cluster at the interface between the hydrocarbon chain and
polar head group region
PREDICTING MEMBRANE SPANNING REGIONS OF INTERGRAL MEMBRANE
PROTEINS
- Membranes spanning regions can be predicted from the amino acid sequence
- Stretches of ~20 hydrophobic residues in a row are likely membrane spanning
- Hydropathy index looks at the hydrophobic characteristics of a protein to predict transmembrane
regions
HYDROGEN BONDING WITHIN MEMBRANR-SPANNING REGIONS
- Side chains within the transmembrane region tend to be the non-polar
o Carbonyl and amide groups of each peptide bond are polar
- Polar unpaired carbonyl and amide groups in the bilayer core are energetically unfavorable
- Carbonyl and amide groups of the protein backbone within the bilayer have to be hydrogen
bonded
TRANSPORT ACROSS MEMBRANES
- Categories of membrane transport
o Simple diffusion
o Facilitated diffusion
▪ Carriers
▪ Channels
o Active transport
▪ Primary
▪ Secondary
o Ion transporters
SIMPLE DIFFUSION
- Non-polar gases (O2 and CO2) and hydrophobic molecules can directly cross membrane
- Direction and rate of movement determined by their concentrations on either side of the
membrane
- Diffusion can only result in the net movement down a concentration
FACILITATED DIFFUSION
- Membrane transporters lower the activation energy barrier of crossing the bilayer
- Activation energy for removing the hydration shell from a polar solute and transferring it into the
non-polar environment in the core of the bilayer is very high
- Membrane transporters lower the activation energy for crossing the membrane for crossing the
membrane by replacing the hydration shell with interactions with polar groups along the transfer
path in the protein interior
FACILITATED DIFFUSION (CHANNELS VS CARRIERS)
- Channels
o Membrane pores to transport molecules down conc gradient
o High conductance rates because they bind the substrate very weakly
o Do not saturate
- Carriers
o Membrane proteins that undergo substrate-induced conformational change, or membrane
repositioning, to release substrate to the other side pf the membrane
o Slower because they bind the substrate quite strongly
o Can saturate
FACILITATED DIFFUSION THROUGH A CARRIER GLUCOSE PERMEASE OF
ERYTHROCYTES
- Facilitated diffusion of glucose at 50,000 X faster than simple diffusion
- Specific for D-glucose
- Rate of uptake follows a pattern resembling M-M kinetics
- Kt about 1/3 the concentration of blood glucose so the transporter is nearly saturated and operates
near Vmax
COUPLED TRANSPORT
- Transport of a single molecule is called uniport
- Some transporters couple the movement of two molecules
o Antitransporters move molecules in different directions
o Symporters move molecules in the same directions
 - During diffusion, co-transport through antiport or symport depends on the charge of the
molecules in order to have a net neutral change
- During secondary active co-transport, system couples a molecule moving down its gradient to one
moving down its gradient
ACTIVE TRANSPORT
PRIMARY ACTIVE TRANSPORT P-TYPE ATPASE
- Input of energy allows movement of molecules against concentration gradients
- Primary active transport
o Driven by direct source of energy (ATP)
o Includes P-type, V-type, and ABC transporters
- Secondary active transport
o Couples the movement of one molecule down its concentration gradient within the
movement of another molecule down its gradient
ACTIVE TRANSPORT (V-TYPE ATPASE AND ABC TRANSPORTERS)
- Cells maintain high gradients of Na+ outside the cell and the K+ inside the cell
- Gradient controls cell volume, electrical excitability, and enables uptake of nutrients through
secondary active transport system
- Maintaining activity the Na+ - K+ pump requires about a third of your energy
- Na+, K+ ATPase use the energy of ATP hydrolysis to pump three Na+ out of the cell and two K+
into the cell
- Called a P-type transporter as in undergoes a phosphorylated intermediate
SECONDARY ACTIVE TRANSPORT (GLUCOSE UPTAKE INTO INTESTINAL EPITHELIAL
CELLS)
- V-type ATPase
o Use the energy of ATP to move protons against the concentration gradient
o Acidification of organelles
o In chloroplasts and mitochondria, F-type ATP synthases reverse this reaction to use
proton gradients to generate ATP
- ABC Transporters
o Contain ATP-binding domains (ATP-Binding cassette)
o Transport of a variety of biomolecules out of the cell against a concentration gradient
o Multi-drug resistance protein pumps drugs (chemotherapeutic) out of the cell rendering
the drugs ineffective
- In intestinal epithelial cells, glucose uptake from the gut is driven through symport with Na+
- The movement of glucose up its concentration gradient is enabled by the movement of Na+ ions
down their concentration
- Active transport of glucose from the gut depends on the action of the Na+ - K+ ATPase to
establish the gradient of Na+ ions
ION CHANNELS
- ion channels enable rapid movement of ions across the membrane
- action of ion channels can cause changes in membrane potential (action potentials) in neurons
- ion channels are tightly regulated; voltage-gated channels and ligand-gated channels
- ion channels differ from ion transporters (N+, K+ ATPase) in three ways
o faster
o no saturations limit
o gated/regulated (open and close in repones to signal)
- ion channels are highly selective for the molecule to be transported
SPCIFICITY OF ION CHANNELS (K+ CHANNELS)
- K+ channels allow rapid movement of K+ ions out of cell
- Although Na is smaller the channel is 100-fold more permeable to K
 -Selectivity filter discriminates K and Na based on their ability to shed water molecules to form
electrostatic interactions within backbone carbonyls
SPEED OF ION CHANNELS (K+ CHANNELS)
- Selectivity filter has four equivalent binding sites for K+
- As K+ ions enter the filter, the electrostatic repulsion from other incoming K+ ions helps to push
the flow of ions form inside to outside the cell
PHYSIOLOGICAL ROLES OF NUCLEOTIDES AND NUCLEIC ACIDS
- Nucleotides
o Energy currency (ATP)
o Signalling molecules (cAMP)
o Enzyme co-factors (NAD, FAD)
o Building blocks of nucleic acids
- Nucleic acids
o Genetic information (DNA, RNA)
o All stages of protein synthesis (DNA,, mRNA, tRNA, rRNA)
STRUCTURAL FEATURES OF NUCLEOTIDES
- Nucleotides are building blocks of nucleic acids
- Nucleotides all share three components
o Ribose sugar (ribose or deoxyribose)
o Nitrogenous base (purine or pyrimidine)
o Phosphates
RIBOSE AND DEOXYRIBOSE
- All nucleotides contain a ribose backbone
- ribose within nucleotides is in a cyclized form (beta-D-ribofuranose)
- for DNA, 2’ carbon of the ribose is the deoxy form
- RNA contains ribose; DNA contains deoxyribose
NITROGENOUS BASES
- Two families of nitrogenous bases
o Purines and pyrimidines
▪ Pyrimidines have a single ring
▪ Purines have two ring system
- Nitrogenous bases are planar and relatively non-polar
- Five standard nitrogenous bases
o Adenine, guanine and cytosine in both DNA and RNA
o Fourth bases differ
▪ DNA has thymine
▪ RNA has uracil
NITROGENOUS BASES
- Nitrogenous bases link to ribose through N-glycosidic bonds
- All the nitrogenous bases link to C1’ of the sugar
- In purines, N-glycosidic bond is to N9 of nitrogenous base
- In pyrimidines, N-glycosidic bond is to N1 of nitrogenous base
PHOSPHATES: NUCLEOTIDES VS NUCLEOSIDES
- Nucleotides and nucleosides differ in whether they are phosphorylated at the C5’ position
- Nucleotides have 1-3 phosphates on the 5’ position: one (NMO), two (NDP), three (NTP)
- Nucleotides are phosphorylated nucleosides
- Nucleosides are unphosphorylated
- Nucleotides are phosphorylated
NOMENCLATURE OF NUCLEOSIDES AND NUCLEOTIDES
- Three things to look at
o Which nitrogenous base is present (base name)
 o Whether the sugar is ribose or deoxyribose (deoxy prefix)
o Whether there are phosphoryl groups (suffix of osine from nucleosides; ylate for
nucleotides)
- Alternate method for naming nucleotides is to specify the number and position of the phosphoryl
groups
PHYSIOLOGICAL ROLES OF NUCLEOTIDES
- Energy transfer
o Anhydride linkages in ATP are high energy bonds
o Energy released from hydrolysis of these bonds drives many biochemical reactions
- Signal transduction
o Cyclic AMP, formed from ATP in a reaction catalyzed by adenylyl cyclase
o Common intracellular messenger produced in response to hormones
PHOSPHODIESTER BONDS JOIN NUCLEOTIDES IN NUCLEIC ACIDS
- Nucleotides from linear nucleic strands through 3’-5’ phosphodiester linkages
- 3’-5’ phosphodiester bonds are identical in DNA and RNA
- 3’-5’ phosphodiester linkages are identical, independent of the nucleotides being joined
- The strand of sugars linked by phosphodiester bridges is called the backbone of nucleic acid
SEQUENCE INFORMATION WITHIN NUCLEIC ACIDS
- Sequence of bases that uniquely characterizes a nucleic acid
- Nucleic acid strands have a direction and their sequences are presented 5’→3’
- Sequences of bases is a form of linear information
RIBONUCLEIC ACID (RNA): STRUCTURE
- RNA differs from DNA in that
o RNA contains ribose rather than deoxyribose
o RNA contains uracil rather than thymine
- RNA is a single-stranded but can adopt complex three-dimensional structures
RIBONUCLEIC ACID (RNA): FUNCTIONS
- Ribosomal RNA (rRNA)
o Integral part of ribosomes
o ~80% of RNA in cells
- Transfer RNA (tRNA)
o Carry activated amino acids to ribosomes for protein synthesis
o Small molecules 73-95 nucleotides long
- Messenger RNA (mRNA)
o Code for proteins
o Contains triplet codons that specify the amino acid sequence of a protein
- Micro RNA (miRNA)
o Short oligonucleotides (22-24 nts in length) that function in transcriptional and post-
translational regulation of gene expression
DIFFERENTIAL STABILITIES OF DNA AND RNA
- 2’ hydroxyl group of RNA increases its susceptibility to base hydrolysis at the phosphodiester
linkages
- Greater stability of DNA is consistent with its role as a long term information storage molecule
DISCOVERING THE DOUBLE HELIX
- James Watson and Francis Crick postulated the double helix structure of DNA in 1953
- Model explained all the known experimental data and predicted mechanism for storing and
replicating the genetic information
- Rosalind Franklin and Maurice Wilkins obtained the x-ray diffraction data that showed that DNA
is a helix
- Efforts set the stage for Watson and Crick
- Watson, Crick and Wilson shared the Nobel prize in 1962
BASICS OF THE DOUBLE HELIX
- Two helical DNA strands coiled around a common axis forming a right-handed double helix
- Strands run in opposite directions
- Strands are complimentary to each other
- Sugar-phosphate backbones are on the outside of the helix, nitrogenous bases on the inside
- Base pairs are perpendicular to helix axis
- Watson-Crick base paring matches a purine with a pyrimidine to give a constant helix diameter

BASE PAIRING IN THE DOUBLE HELIX

- Adenine (A) base pairs with Thymine (T)
- Guanine (G) base pairs with Cytosine (C)
- Specificity of Watson-Crick base pairing is largely determined by the hydrogen bonding groups
of the nitrogenous bases
- Chargaff’s rule: A+G=T+C
- Number of purines equals the number of pyrimidines in duplex DNA
- A-T and G-C hydrogen bonded pairs are planar and have the same directions
WEAK FORCES STABILIZE THE DOUBLE HELIX
- Hydrophobic effects
o Burying purine and pyrimidine rings in the interior of the helix
- Stacking interactions
o Stacked base pairs form Van Der Waals contacts
- Charge-charge interactions
o Electrostatic repulsion of negatively charged phosphate groups is decreased by cations
and cationic proteins
MAJOR AND MINOR GROOVES
- Many proteins bind DNA in a sequence-specific fashion
o Restriction enzymes
o Transcription
- Double helix has two grooves of unequal width
o Major groove
o Minor groove
- Within each groove base pairs are exposed and are accessible to interactions with other molecules
- DNA-binding proteins can use these interactions to “read” a specific sequence
RESTRICTION ENDONUCLEASES
- Restriction endonucleases recognize and cleave specific DNA sequence
- Bacterial defense mechanism against viral invasion
- Host cells protect their own DNA by covalent modification of bases at the restriction site
o Methylation
- Names of restriction enzymes reflect origins
o bamHI is the first restriction enzyme characterized from Bacillus amyloliquefaciens
strain H
o EcoRV is the fifth restriction enzyme characterized from Escherichia coli R
- Restriction enzyme cut at palindrome sequence
- Restriction enzymes can be used as “molecular scissors” for manipulation of DNA
DNA FINGERPRINTS
- Treating DNA from different individuals with restriction enzymes will break DNA into pieces
- Due to differences in genome sequences, DNA from different people will break down into a
different number of fragments and fragments of different sizes
- Highly variable regions give restriction fragments that are as unique as fingerprints and can be
used to identify individuals in a larger population
- Restriction fragments length polymorphisms (RFLP)
COMPLIMENTARY STRANDS IN DUPLEX DNA
- Duplex DNA contains two complimentary, anti-parallel strands
- Strands are complimentary
o The sequence of one strand determines the sequence of the other strand
DNA AS A CARRIER OF GENETIC INFORMATION
- Complimentary nature of the strands is important for replication and repair
- As the nucleotide sequence of one strand determines the sequence of the other
o Each strand can be used as a template to produce the other
- The resulting two DNA duplexes will be identical
DENATURATION OF DNA
- Denaturation
o Complete separation of double-stranded DNA by heat or chemical agents
- Denaturation of DNA is a cooperative process
- Annealing
o Reforming the double-stranded helix from single strands
- Melting point (T)
o Temperature at which half the DNA has become single stranded
- Melting temperature reflect sequence composition
o Higher the GC content the higher the Tm
SYNTHESIS OF NUCLEIC ACIDS
- DNA and RNA polymerases are the primary enzyme for synthesizing nucleic acids
- Nucleotide triphosphate are the substrates for synthesis
- All polymerase synthesize nucleic acids in the 5’ to 3’ direction
- Incoming residues are added to the 3’ end of the growing strand
- Incoming residues are selected to be complimentary to the template strand
POLYMERASE CHAIN REACTION (PCR)
- Polymerase chain reaction (PCR) takes advantage of the ability for each DNA strand to serve as a
template for production of a complimentary strand
- Uses heat stable enzymes to make new DNA
- Allows for exponential amplification of short regions of DNA very quickly
- PCR revolutionized molecular biology, diagnostics and forensics
- Discovered by Kary Mullis and resulted in a Nobel prize
PACKAGING OF EUKARYOTIC DNA
- Amount of eukaryotic DNA necessitates its packaging into higher order structures
- First level of DNA packaging involves formation of nucleosomes
- Nucleosomes “beads” are DNA-histone complexes on a “string” of double stranded DNA
EUKARYOTIC DNA IS PACKAGED IN NUCLEOSOMES
- Histones are DNA packaging proteins
- Histones are highly conserved and positively charged
- Five histone proteins
o H1
o H2A
o H2B
o H3
o H4
- Nucleosome composed of two molecules of each H2A, H2B, H3, H4 AND 146 base pairs of
DNA
- H1 binds the region of linker DNA
- Histones are reversibly modified to regulate their interaction with DNA
SOME BASICS OF GENETIC INFORMATION
 - Gene is a segment of DNA containing the information for production of a functional biological
product (like protein)
- Size of gene may be estimated from the size of the corresponding protein
- 3 nucleotides = 1 codon = 1 amino acid
- Genes are contained within chromosomes
- Viruses and bacteria have single chromosomes
- Eukaryotes have multiple chromosomes
BACTERIAL GENOME
- Millions of based pairs
- Closed, circular genome
- No internal interruptions (introns)
- Bacteria may have additional genetic info in the form of plasmids
o Plasmids are non-chromosomal DNA
o Many plasmids encode information for resistance to antibiotics
o Plasmids may be isolated and manipulated
EUKARYOTIC GENOME
- Billions of nucleotides divided among numerous chromosomes
- Different organisms have different numbers of chromosomes
- Each chromosomes have a characteristic set of genes
- Eukaryotic chromosomes are linear, which presents a problem for replicating the ends of
chromosomes
o Ends of chromosomes containing repeating sequences called telomeres
- Genes interrupted by non-coding regions
o Introns
- Some organelles may contain additional DNA distinct from that of the nucleus
o Mitochondria and chloroplasts
COMPLEXITY OF EUKARYOTIC GENOME: INTRONS
- Most eukaryotic genes interrupted by non-coding intervening sequences (introns)
- Exons contain protein-coding information
- Introns may vary in size, number and position
- Introns removed from mRNA prior to translation
- One functional advantage of introns is that multiple mRNAs of different sequences can be
generated from a single gene
EPIGENETICS
- Conventional genetics suggests individuals inherit genetic material which they pass to their
offspring
o That we are carriers rather than editors of genetic info
- Environmental influences our genetic material can covalently modified
- Such modifications include methylation of cytosine residues
- These modifications of DNA can be heritable
o Passed to offspring
- Epigenetics refers to functionally relevant changes to the genome that do not involve a change in
the nucleotide sequence
- Epigenetic changes can alter patterns of gene expression without altering the underlying DNA
sequence
MOLECULAR BIOLOGY AND THE MODERN WORLD
- Advances in sequencing technology and computer science position us to better understand the
blueprint of life
- Comparing genomes from different species, as well as individuals from within species, can
provide insight into phenotypic differences
- Genomic approaches enable better prediction, understanding, and treatment of disease
 o Personalized medicine
- Genetic manipulation also allows creation of novel organisms with desirable traits
o GMO crops
o Low fat pigs
o Glow-in-the-dark pets