U of S BIOC200 - Biomolecules Final Notes

05/12/08 11:07:49 AMmidterm notes.oo3

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I. Introduction to biomoleculesA. Functional groups

1. hydroxyla) R-OH

2. carbonyla) R-CO-Rb) R-CO-H

3. carboxyla) R-COOH

4. Ethera) R-O-R

5. Estera) R-COO-R

6. sulfhydryl (thiol)a) R-SH

7. disulfidea) R-SS-R

8. thioestera) R-COSH

9. phosphoryl

a) R-POO--OH10. amino

a) R-N-RR11. amide (amido)

a) R-CO-NHRB. Isomers

1. structurala) differ in bonding sequence

2. stereoisomersa) diastereomer

(1) non mirror imagesb) enantiomers

(1) non-superimposable mirror images(2) optically active(3) distinguished by:

i) D /L(a) configuration relative to glyceraldehyde(b) if OH group farthest from top faces right when most oxidized group at top (facing away), then D configuration

ii) R/S(a) absolute configuration by priority of groups bonded to chiral centre

iii) +/-(a) direction of rotation of polarized light

C. Structure of biomolecules1. the 3D arrangement in space

a) important for biological activity(1) binding substrate to enzyme

b) described by:(1) configuration

i) spatial arrangement about double bonds or chiral centresii) can only be changed by breaking bonds

iii) E/Z, R/S(2) conformation

i) spatial arrangement of groups that are free to assume different positions without breaking bondsii) chair/boat in cyclohexane

2. reactivity of biomoleculesa) result of their functional groups


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I. Introduction to biomolecules

C. Structure of biomolecules

2. reactivity of biomoleculesa) result of their functional groupsb) oxidation

(1) loss of electrons, H or addition of O(2) increased CO, CN bonds; decreased CH

c) reduction(1) gain of electrons or H, or removal of O(2) increased CH bonds; decreased CO, CN

D. Gibb's free energy1. the useful work that may be obtained from a thermodynamic system2. the change in Gibb's energy (∆G) determines the spontaneity of a given reaction

a) ∆G<0 spontaneous, will proceed until equilibrium is reached, exergonicb) ∆G>0 non spontaneous, required input of energy to proceed, endergonicc) ∆G=0 reaction at equilibrium, no change in free energy

3. in biological systems, non-spontaneous reactions are coupled to spontaneous reactions so that they may occura) possible since ∆G values for the individual reactions in a pathway are additive

4. ∆G° is the standard change in gibbs energy for a given reaction at [1M] of reactants and products at 298K (25°C)a) ∆G=∆G°+RT ln([Products]/[Reactants])

(1) R=molar gas constant(2) T=absolute temperature

b) at equilibrium

(1) ∆G°=–RT ln(Keq)

5. Overall ∆G is related to entropy and enthalpya) entropy (∆H)

(1) change in heat of reactionb) enthalpy (∆S)

(1) change in randomness of systemc) ∆G=∆H-T∆S

II. WaterA. General Properties

1. polar moleculea) electronegativity difference between O and H creates dipoles

2. 70% of humans by mass3. polarity causes cohesion (H-bonding) and adhesion (dipole-dipole)4. limited dissociation

B. H-bonding1. weak electrostatic attraction between lone pair electrons of O or N (electronegative) and H attached to another electronegative atom

2. H-bonds of short duration (10-12 s)a) form and break constantlyb) 3.4 H-bonds at a time in liquid phase, 4 in solid

3. gives water high boiling point and heat of vapourizationC. Solvent properties

1. good solvent of polar moleculesa) orients itself to maximize electrostatic attraction to other moleculesb) in solvation, charged particles are surrounded or insulated by water molecules

2. Interactions with biomoleculesa) H-bonding with polar and ionic biomoleculesb) solubility increases with number of polar groupsc) influences amphipathic molecules

(1) amphipathici) molecule with polar group and non-polar region

(a) fatty acids(2) causes formation of aggregates

i) micelles, bilayersii) stablilized by H-bonding and hydrophobic tendency to minimize interaction between non-polar regions and water

3. Non-covalent interactions in biomolecules


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II. Water

C. Solvent properties

2. Interactions with biomolecules

c) influences amphipathic molecules

(2) causes formation of aggregates

ii) stablilized by H-bonding and hydrophobic tendency to minimize interaction between non-polar regions and water3. Non-covalent interactions in biomolecules

a) critical to structure and function of biomoleculesb) ionic

(1) electrostatic interactions between charged moleculesc) hydrogen bonds

(1) weak electrostatic interactions between lone pair electrons and H atomsd) Van der Waals forces

(1) non-covalent attractive forces between 2 neutral molecules due to dipolesi) dipole-dipole(a) attraction between 2 permanent dipoles

ii) dipole-induced dipole(a) a transient dipole is induced by a nearby dipole by random attraction of electrons

iii) induced-induced dipoles(a) transient dipoles formed for very short periods due to random, uneven distribution of electrons

e) hydrophobic(1) association of non-polar molecules in aqueous solution rather than with water

D. Dissociation of water

1. 2 H2O ⇌ H3O+ + OH-

a) Water acts as both a Bronsted-Lowry acid and bsase (amphoteric)2. dissociation is limited

3. Kw = [H3O+][OH-] = 10-14 M

4. pH = -log [H+]E. Weak acids and bases

1. maintain pH in living orgnisms2. partial ionization allows creation of buffer solutions

a) resist changes in pH by increasing or decreasing ionization due to Le Chatelier's principle(1) unlike strong acids which ionize completely in solution

3. Henderson Hasselbalch Equation

a) represents the pH of a buffer solution as OH- is added

b) pH=pKa+ log([A-]/[HA])

c) buffer solution is best able to resist pH change when HA is half-ionized

(1) pH=pKa

(2) most buffers have a buffering zone of ± 1 pH on each side of pKad) polyprotic acids will have more than one buffering zone

III. CarbohydratesA. General

1. typesa) monosaccharides

(1) simple sugars(2) one sugar(3) single polyhydroxyaldehyde/ketone(4) glucose, fructose, etc.

b) oligosaccharides(1) short chains of monosaccharides linked by glycoside bonds(2) disaccharides:sucrose,lactose

c) polysaccharides(1) linear or branched chains of monosaccharides(2) can be linked to lipids or proteins(3) starch, cellulose

2. function:a) primary energy source

(1) oxidized to CO2 + H2O + ATPb) storage form of energy


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III. CarbohydratesA. General

2. function:a) primary energy source

(1) oxidized to CO2 + H2O + ATPb) storage form of energy

(1) Glycogen, starchc) biosynthesisd) structural components

(1) cellulose(2) chitin

e) informational roles(1) recognition complexes

B. Monosaccharides1. polyhydroxy aldehyde = aldose

a) glucoseb) most have -ose suffixc) smallest is glyceraldehyde

2. polyhydroxy ketone = ketosea) suffix -uloseb) smallest is dihydroxyacetone

3. Most monosaccharides are in D configuration4. epimers

a) monosaccharides which differ only in configuration at one chiral carbon(1) glucose / galactose @ C4

5. monosaccharides can exist in a ring form by forming a hemiacetal or hemiketal by bonding to carboxyl carbon (anomeric)a) anomers

(1) cyclic monosaccharides can exist in two configurations of the straight chain form(2) α: OH group on anomeric carbon E to other group on ring, usually C6(3) ß: OH group on anomeric carbon Z to other group on ring

b) anomers interconvert using straight form as intermediatec) 5 membered rings:

(1) furanosed) 6 membered rings:

(1) pyranose(2) has boat and chair conformations (chair most stable)

i) glucose most stable monosaccharide since it has all non-H substituents in equatorial position of chair conformatione) if a group is covalently linked to the anomeric carbon's OH, it constitutes a glycosidic bond

6. reducing sugarsa) a sugar is reducing if it has a free carboxyl group in the straight-chain formb) all monosaccharides are reducing, but not all oligosaccharides

(1) lactose is reducing since it's 1-4 glycosidic bond only occupies one of the two carboxyl groups(2) sucrose is not reducing because it's 1-2 glycosidic bond occupies both carboxyl groups (bond is to the anomeric carbon of each

constituent monosaccharide)c) if aldehyde or ketone is reduced to an alcohol, it results in sugar alcohols

(1) glucose -> sorbitol7. Examples

a) mannose(1) hexoaldose(2) pyranose(3) configuration of highest priority groups after anomeric carbon (in numerical order)

i) up-up-down

(4)

b) glucose(1) hexoaldose (anomeric carbon C1)(2) pyranose


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III. Carbohydrates

B. Monosaccharides

7. Examples

b) glucose(1) hexoaldose (anomeric carbon C1)(2) pyranose(3) configuration of highest priority groups after anomeric carbon (in numerical order)

i) down-up-down

(4)

c) galactose(1) hexoaldose(2) pyranose(3) configuration of highest priority groups after anomeric carbon (in numerical order)

i) down-up-up

(4)

d) fructose(1) hexoketose (anomeric carbon C2)(2) furanose(3) configuration of highest priority groups after anomeric carbon (in numerical order)

i) up-down-up (group on C5 is CH2OH)

(4)

C. Oligosaccharides1. 2-20 monosaccharides linked via glycosidic bonds2. disaccharides formed when two monosaccharides bond by the OH on anomeric carbon of one monosaccharide forms an acetal with an OH on

the second monosaccharide by removing an HOH molecule (dehydration synthesis)3. α or ß configuration of sugar giving anomeric OH determines configuration of resulting glycosidic bond4. if the chain formed has a free anomeric carbon, that end is called the reducing end (see B.6)5. oligosaccharides named from non-reducing end towards the reducing end6. anomeric configuration of reducing end may interconvert7. Examples

a) maltose(1) α-D-glucopyranosyl-(1–4)-ß-D-glucopyranose

i) glycoside bond is αii) sugar is reducing (1–4 bond)

iii) -syl means the molecule is inside the chainiv) -ose means the end of the chain is reducing

v)

b) lactose


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III. Carbohydrates

C. Oligosaccharides

7. Examplesa) maltose

(1) α-D-glucopyranosyl-(1–4)-ß-D-glucopyranose

v)

b) lactose(1) ß-D-galactopyranosyl-(1–4)-ß-D-glucopyranose

i) glycoside bond is ßii) sugar is reducing

iii)

c) sucrose(1) α-D-glucopyransyl-( 1–2)-ß-D-fructofuranoside

i) glycoside bond is αii) sugar is non-reducing (both anomeric carbons involved in bonding)

iii) -oside means that fructose has no reducing end

iv)

D. Polysaccharides1. >20 sugars2. Classification

a) homopolysaccharides(1) same sugar residues

i) amylose (D-glucose)b) heteropolysaccharides

(1) different sugar residues3. Starch

a) amylose(1) unbranched glucose polymer

i) α1–4 bondsii) 10-1000 residue chains

(a) single chains coil into tight helices(2) storage form of carbohydrate in plants and fungi(3) broken down by α-amylase

i) cleaves 1–4 bond between glucosesb) amylopectin

(1) branched glucose polymer(2) main chain segments are α1–4 bonds, branches attached with α1–6 bonds

i) 300-600 residuesii) branches create many non-reducing ends

iii) branched every 20-25 residues(a) prevents helix formation

(3) broken down by α-amylase (1–4) and debranching enzyme (1–6)c) glycogen

(1) more highly branched form of amylopectini) every 8-10 residues

(2) used as animal storage carbohydrate


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III. Carbohydrates

D. Polysaccharides

3. Starch

c) glycogen(1) more highly branched form of amylopectin

i) every 8-10 residues(2) used as animal storage carbohydrate

i) stored in liver and muscle cells(3) very high number of non-reducing ends allows rapid release of glucose into bloodstream

d) cellulose(1) structural component of plants(2) up to 15 000 glucose molecules

i) ß1–4 linked with extensive H-bonding(a) results in very strong and insoluble structure

(3) ß-glucosidase (cellulase) cleaves ß1–4 bonds

IV. Organic Chemistry ReviewA. Reactions

1. oxidationa) R-OH —> R=Ob) primary alcohol —> aldehyde —> carboxylic acidc) secondary alcohol —> ketone

2. esterificationa) alcohol/thiol + carboxylic acid —> ester/thioester + HOH

3. reductiona) aldehyde —[H+]—> primary alcoholb) ketone —[H+]—> secondary alcoholc) carboxylic acid —[H]—> aldehyde

4. hemiacetal formationa) aldehyde + alcohol <—> hemiacetal

5. acetal formationa) hemiacetal + alcohol —> acetal + HOH

6. aldol condensationa) 2 aldehydes or ketones —> hydroxyadehyde/ketone

7. acid anhydridea) 2 carboxylic acids —> acid anhydride

8. amide formationa) carboxylic acid + ammonia/amine —> amide + HOH

V. 3 Dimensional Structure of ProteinsA. Physiological Roles of Proteins

1. Enzymesa) biochemical catalysts

2. Storage and transport3. Cellular structure4. Mechanical movement5. Decoding cell information6. Hormones or hormone receptors7. Other specialized functions8. Each protein has specialized structure for specific function

B. General Classes of Proteins1. Fibrous

a) simple, linear structuresb) structural

2. Globulara) spherical and solubleb) enzymes and transport

3. Membranea) associated with a membraneb) receptors, transporters, pumps

C. Protein Conformation and Folding1. native proteins only marginally stable2. stabilized by weak interactions

a) greatest number of weak interactions is the most stable conformation (lowest ∆G)


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V. 3 Dimensional Structure of Proteins

C. Protein Conformation and Folding

2. stabilized by weak interactionsa) greatest number of weak interactions is the most stable conformation (lowest ∆G)

3. native conformation is the single stable folding shape at physiological conditions4. Folding

a) rapid step-wise process (<1 second)b) individual secondary structures first, then core condenses to tertiaryc) some protein folding spontaneous, others require chaperone proteins

5. Denaturationa) disruption of native conformation; loss of biological functionb) energy required only 3-4 H-bond equivalentsc) cooperative processd) only some proteins can renature

D. 4 Levels of Protein Structure1. Primary

a) linear sequence of amino acids(1) no three-dimensional structure

b) defines composition of amino acidc) sequence presented from N –> C termina

(1) eg. NH3+–MLCDTN–COO-(2) repeating pattern of N-CR-C-N-CR-C-N in main chain

2. Secondarya) localized interactions within polypeptideb) patterns in local conformation due to H-bonds between amide H's and carbonyl O'sc) a region of secondary structure will have a single H-bonding pattern

(1) polypeptide folding is restricted by limited flexibility of peptide bondd) determinants of allowed secondary structures

(1) favoured conformations of peptide bondi) CN bond exhibits resonance, restricting rotation

(a) rotation only about N-CR (Phi Φ) and CR-C (Psi Ψ) bondsi) most conformations prevented by steric interference

ii) allowed conformations graphed on Ramachadran plotii) almost all peptide bonds in proteins are trans (E)

(2) optimization of hydrogen bonding potentiali) each peptide bond has both H-bond donor and acceptor

(a) donor: NH group(b) acceptor: C=O group

e) Structures:(1) α-helix

i) right-handedii) 3.6 residues/turn(a) H-bonding between C=O and the H-N 4 residues away

iii) stabilized by many H-bonds parallel to axis of helixiv) entire helix is a dipole

(a) + at N terminus(b) - at C terminus(c) all C=O groups point toward C terminus, all NH groups point toward N terminus(d) dipole communicated to ends through H-bonding(e) primary structure can stabilize helix by having positive residues at C terminus and negative at N terminus

v) Φ and Ψ angles of each residue similarvi) primary structure affects helix stability

(a) many positive or negative residues in sequence usually do not form helices due to electrostatic repulsion(b) residues 3-4 positions apart will be close together in helix

i) positive usually found 3-4 positions from negative residues(c) proline seldom found in helices due to rigidity (known as helix-breaker)

vii) Amphipathic helix(a) helix in which hydrophobic residues are on one side of the helix (all 3-4 residues away in primary structure) and hydrophilic on the

opposite side


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D. 4 Levels of Protein Structure

2. Secondary

e) Structures:(1) α-helix

vii) Amphipathic helix(a) helix in which hydrophobic residues are on one side of the helix (all 3-4 residues away in primary structure) and hydrophilic on the

opposite side(2) ß-strands and sheets

i) ß-strand(a) polypeptide chains almost fully extended

ii) ß-sheet(a) multiple ß-strands arranged side by side with H-bonds between(b) parallel ß-sheets

i) strands run in the same N –> C termina direction(c) antiparallel ß-sheets

i) stands run in alternate N–>C termina directionsii) H-bonding more stable; more perpendicular to chains

(A) antiparallel slightly more stable than parallel(d) residue R groups project alternately above and below sheet

i) every second R group on the same sideii) amphipathic ß-sheets have one hydrophobic and one hydrophilic side

(A) can interact with amphipathic α-helices(3) Turns

i) connect helices and strandsii) allow peptide chain to fold back on itself

iii) are loops with < 5 residuesiv) ß-turns (reverse turns)

(a) connect different antiparallel ß-strands3. Tertiary

a) final structure of a single polypeptide4. Quaternary

a) structure involving multiple polypeptides(1) multiple subunits joined together

i) may be same type or different polypeptides(2) held by non-covalent interactions

b) helps stabilize subunits; prolongs life of proteinc) unique active sites at subunit interfacesd) unique/dynamic function through changes in tertiary and quaternary structuree) combinations of simpler subunits more efficient than selection for new protein with ideal function

E. Examples of Protein Structure/Function1. Keratin

a) 2 RH α-helices twisted into LH coil(1) α-helices associate by hydrophobic interactions

i) each helix has hydrophobic side due to a 7-residue pseudorepeat of hydrophobic residuesb) stabilized by disulfide bonds

(1) more disulfides, greater hardnessi) hair vs. horn

c) forms hair, nails, horns2. Collagen

a) 25% of human protein(1) major structural protein that forms skin and tendons

b) 3 LH helical chains coiled into RH triple helixc) repeats of Gly-X-Y, where X often Proline or Y often hydroxyproline

(1) Gly R group faces inwards; -H is only group small enough to fit inside(2) hydroxyproline, hydroxylysine

i) formed by hydroxylation reactions with vitamin C cofactorii) stabilize collagen

iii) scurvy leads to defective triple helix(a) humans cannot synthsize vitamin C

3. Silka) cocoons, webs


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E. Examples of Protein Structure/Function

3. Silka) cocoons, websb) very high strengthc) small amino acids in 6 residue repeat

VI. Protein FunctionA. Reversible binding of a protein to a Ligand — Oxygen transport proteins

1. heme prosthetic groupa) non-polypeptideb) found in both myoglobin and hemoglobinc) tetrapyrrole ring bound to iron

(1) iron has 4 bonds to tetrapyrrole ring; 2 free bond locations

(2) in Mb and Hb, one position occupied by histidine, the other is free to bond with O2, CO, or NO

i) neither Mb and Hb, or heme can bind oxygen alone; only when bonded together2. Myoglobin (Mb)

a) Structure(1) monomeric protein

i) globin polypeptide covalently bonded to heme prosthetic groupii) single oxygen binding site

(a) strong reversible interactionb) facilitates oxygen transport in peripheral tissuec) called Oxymyglobin when oxygen-boundd) Deoxymyoglobin is oxygen-freee) Oxygen binding curve is hyperbolic with half saturation point ~ .26 kPa

(1) saturation = [pO2]/([PO2]+P50)

3. Hemoglobina) tetrameric

(1) each globin subunit has own heme groupi) 4 oxygen bonding sites

(2) each subunit similar to single myoglobin proteinb) found in RBCs that transports oxygen from lungs to periphery through blood

(1) 96% sat in arteries, 64% in veins under normal conditionsc) Binding curve

(1) Hb exhibits positive cooperativity

i) O2 affinity increases as more O2 is bound

(a) structural change is initiated when one Hb subunit is boundi) subunits are tight enough that tertiary change in one subunit, changes conformation of the other 3

ii) oxygen causes subunits to go from tense to relaxed structuresd) allosteric properties

(1) Hb is an allosteric proteini) it's activity is affected by allosteric effectors that bind at sites separate from the functional site

(a) allosteric activators stabilize R state, shifting equilibrium in R direction(b) allosteric inhibitors stabilize T state, shifting equilibrium in T direction

ii) 2,3BPG (Bisphospho-D-Glycerate) is an allosteric inhibitor of Hb(a) is a negatively charged molecule that binds to six positive residues at its receptor

i) fetal Hb has only 5 residues(A) is less inhibited in order to take oxygen from maternal Hb by having higher affinity

(b) lowers oxygen affinityi) allows oxygen to be transferred from Hb to Mb

e) Bohr effect

(1) Hb O2 affinity increases with pH

(2) CO2 reduces pH in blood, lowering affinity, releasing O2 into tissue

4. Sickle Cell Anemiaa) Hb mutation of E –> Vb) forms fibres at low partial oxygen pressure which block capillariesc) selective advantage in malaria endemic areas

(1) malaria lowers pH in infected cells, causing fibres and spleen destroys infected cells


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VI. Protein FunctionA. Reversible binding of a protein to a Ligand — Oxygen transport proteins

4. Sickle Cell Anemia

c) selective advantage in malaria endemic areas(1) malaria lowers pH in infected cells, causing fibres and spleen destroys infected cells

VII. EnzymesA. Overview

1. enzymes are required for lifea) efficiently and selectively catalyze chemical reactions required for lifeb) most biomolecules too stable to transform quickly enough for life

2. practical applicationsa) genetic disorders involve enzyme deficiencyb) biomarkersc) drugs designed to influence enzymesd) commercial/industrial

3. most enzmes are proteinsa) some RNA molecules are the exception

4. many enzymes have full activity alonea) others require co-factors (inorganic ions) or co-enzymes (organic/ vitamins)

(1) a tightly associated co-enzyme or co-factor is called a prosthetic groupheme in Hb and Mb

b) Apoenzyme(1) enzyme which does not have a required co-factor/enzyme

c) Holoenzyme(1) Apoenzyme that has aquired a required co-factor/enzyme

B. Classification1. Oxidoreductases

a) transfer of electrons2. Transferases

a) group transfer reactions3. Hydrolases

a) hydrolysis (transfer of functional groups to water)4. Lyases

a) addition of groups to double bondsb) formation of double bonds

5. Isomerasesa) transfer of groups within molecules to get isomeric forms

6. Ligasesa) formation of C-C, C-S, C-O, C-N bonds by condensation with ATP cleavage

C. Function1. lower activation energy of the transition state by using alternate reaction pathway

a) increase rate without changing equilibrium(1) ∆G remains the same, ∆G‡ (activation energy) decreased

b) relationship between rate constant and activation energy is inverse and exponential

(1) rate enhancements can be of the order of 107

2. not consumed by reaction3. compared to chemical catalysts:

a) fasterb) milder conditionsc) greater specificityd) can be regulated

4. catalyse the interconversion of substrate and product5. active site

a) portion of the enzyme that binds the substrate, forming ES complex6. some enzymes limited by diffusion since reaction proceeds faster than encounters between enzyme and substrate

a) circe effect(1) some enzymes even faster than diffusion limits due to electrostatic attraction of substrate molecules

D. Modes of enzymatic catalysis - how activation energy is reduced1. Binding effects

a) substrate binding


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VII. Enzymes

D. Modes of enzymatic catalysis - how activation energy is reduced1. Binding effects

a) substrate binding(1) not only provides substrate specificity, but increases rate by proximity effect

i) enzyme gathers and positions substrates(a) reduces entropy(b) desolvates substrate(c) distorts substrate(d) aligns substrate with enzyme(e) induced fit

b) transition-state stabilization(1) enzyme is complementary to transition state (not substrate)

i) must be similar enough to substrate to be specific, but different enough to promote reaction(2) transition state analogues

i) antibody formed against transition state analogue may act as an enzyme to bind substrate and push towards transition state2. Chemical effects

a) acid/base catalysis(1) polar, ionizable residues act as proton donors/acceptors to catalyse reaction

i) same mechanism as acid/base catalysed reactions in organic chemistryb) covalent catalysis

(1) substrate bonds covalently to enzyme forming reactive intermediate which participates in a second reaction to restore enzyme and form product

E. Enzyme Kinetics1. Velocity

a) V=∆[P]/∆t2. Influencing factors

a) Temperature and pH(1) bell shaped curve about optimum pH/temperature

b) Enzyme concentration(1) linear relationship

3. Initial velocity (V0)

a) velocity at the beginning of reaction; prior to product formationb) dependant on substrate concentrationc) ES —> E + P must be rate-determining step of reaction for M-M approximation

(1) V0=[ES]k2d) steady-state approximation

(1) ES formation=ES breakdowne) Michaelis-Menten equation

(1) V0= {(Vmax)[S]}/{Km+[S]}

i) Vmax= maximum initial velocity

(a) dependant on enzyme concentration/velocityi) enzyme fully saturated with substrate

ii) Km= substrate concentration at 1/2 maximum initial velocity

4. comparison of enzyme activities

a) Kcat=Vmax/[E]t

(1) number of substrate molecules converted to product per unit time in saturating conditions5. Lineweaver-Burk plot

a) double-reciprocal analysis of kinetic data

(1) V0-1=(Km/Vmax)([S]-1) + Vmax

-1

b)


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VII. Enzymes

E. Enzyme Kinetics

5. Lineweaver-Burk plota) double-reciprocal analysis of kinetic data

(1) V0-1=(Km/Vmax)([S]-1) + Vmax

-1

b)

c) Reversible Inhibitors(1) Compound that binds to an enzyme and interferes with its activity

i) non-covalent interactions(2) Competitive

i) Binds to free enzyme E; competes with S for E(a) only either I or S can bind with E at a given time

ii) usually resembles substrate

iii) Vmax is the same; Km is increased

(a) inhibition can be overcome by increasing [S]

iv)

(3) Uncompetitivei) binds to ES, not E

ii) Vmax decreased due to production of ESI

(a) not all ES will immediately form E + P(b) takes longer for P to separate from E

iii) Km decreased

(a) since [ES] decreased due to production of ESI, from LCP, apparent substrate affinity of enzyme increasesi) equilibrium shifts towards ES

iv)


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VII. Enzymes

E. Enzyme Kinetics

5. Lineweaver-Burk plot

c) Reversible Inhibitors

(3) Uncompetitive

iii) Km decreased

(a) since [ES] decreased due to production of ESI, from LCP, apparent substrate affinity of enzyme increasesi) equilibrium shifts towards ES

iv)

(4) Mixed inhibitorsi) binds to both E and ES(a) I does not bind to active site

ii) Vmax decreases due to formation of 2 non-productive complexes EI and ESI

iii) Km does not change

actually, only true if rates of E + I —> EI and ES + I —> ESI are the same, which is non-competitive inhibition. Plot below is of mixed inhibition. By above definition, each line should have the same x-intercept

iv)

d) Irreversible Inhibition(1) inhibitors that form stable covalent bonds with enzyme or disrupt essential functional group of the enzyme(2) Suicidal Inactivators

i) initially unreactive, but after first steps of catalysis, are converted to reactive species that inactivates the enzymeii) form of irreversible inhibition using normal catalytic process

F. Serine Proteases1. cleave peptide bonds in protein substrates2. digestive enzymes

a) trypsinb) chymotrypsinc) elastase

3. covalent and acid-base catalysis4. stored as inactive zymogens that are activated by proteolysis

G. Regulation of Enzymes1. Regulation of availability

a) location, rates of synthesis and degradation2. Regulation of activity

a) covalent modification(1) phosphorylation, methylation, glycosylation(2) interconvertible enzymes(3) catalyzed by converter enzymes


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VII. Enzymes

G. Regulation of Enzymes

2. Regulation of activitya) covalent modification

(2) interconvertible enzymes(3) catalyzed by converter enzymes

i) controlled by allosteric modulatorsb) allosteric modification

(1) binding of a regulatory molecule3. Points of regulation

a) processes usually regulated at rate-limiting step(1) negative feedback inhibition(2) conserves material and prevents accumulation of intermediates

4. Allosteric enzymesa) regulated by interaction with metabolic intermediates

(1) modulatorsi) activators and inhibitors

(a) activators and substrates bind only to the R state(b) inhibitors bind only to T state

(2) bind non-covalentlyb) usually quaternary structuresc) do not obey M-M kinetics; sigmoidal curvesd) PFK-1

(1) Activated by ADP(2) inhibited by PEP

VIII. LipidsA. General

1. hydrophobic2. low molecular weight relative to nucleic acids and proteins

B. Biological Functions1. Energy storage2. Structural

a) cell membranes3. biochemical signals

a) steroid hormones4. enzyme cofactors5. pigments

C. Triacylglycerols (Triglycerides)

1.

2. storage lipids in plants and animals3. glycerol with 3 fatty acids bonded by ester bonds

a) each fatty acid usually different(1) even number of atoms(2) between 12-24 carbon atoms long

b) saturated fatty acids have no double bondsc) unsaturated have 1-3 double bonds

(1) conjugated double bonds rare4. melting point

a) related to intermolecular forcesb) increases with length of fatty acid chainsc) decreases with number of double bonds

(1) kinks in chains make less efficient packing; lower IMF5. Fatty acid numeric nomenclature:


16

VIII. Lipids

C. Triacylglycerols (Triglycerides)

4. melting point

c) decreases with number of double bonds(1) kinks in chains make less efficient packing; lower IMF

5. Fatty acid numeric nomenclature:

a) 18:2(∆9,12)(1) 18 Carbon(2) 2 double bonds at C9 and C12

D. Lipids versus Carbohydrates1. Lipids store more energy, but slower to release2. Lipids release water when broken down, polysaccharides require water to break down

E. Structural Lipids1. form cell membranes that separate aqueous compartments2. 2 sets of amphipathic lipids with polar headgroups and hydrophobic tails form bilayers

a) composed of:(1) backbone(2) hydrocarbon tails; fatty acids(3) polar headgroup(4) Examples

i) phospholipids(a) glycerophospholipids

i) glycerol backboneii) PO4+alcohol head

(b) sphingophospholipidsi) sphingosine backbone

ii) PO4+choline or other headii) glycolipids

(a) Sphingoglycolipidsi) sphingosine backbone

ii) mono or oligosaccharide head(b) Galactolipids (sulfolipids)

i) glycerol backboneii) mono or disaccharide and SO4 head

iii)

3. Glycerophospholipidsa) usually in animal cellsb) glycerol backbonec) C1 usually saturated fatty acid 16-18d) C2 usually longer unsaturated 18-20e) C3 Phosphatditic acid derivative

(1) PO4 group with phosphodiester bond to head group constituenti) Phosphaditic acid(a) -H(b) -1 charge

PO4 group has 1- charge + charge of head group

ii) Phosphatidylethanolamine


17

VIII. Lipids

E. Structural Lipids

3. Glycerophospholipids

e) C3 Phosphatditic acid derivative(1) PO4 group with phosphodiester bond to head group constituent

i) Phosphaditic acid

(b) -1 chargePO4 group has 1- charge + charge of head group

ii) Phosphatidylethanolamine

(a) -CH2-CH2-N+H3(b) OH from alcohol forms part of PO4 group(c) 0 charge

iii) Phosphatidylcholine

(a) -CH2-CH2-N+(CH3)3(b) 0 charge

iv) Phosphatidylserine

(a) -CH2-CH(COO–)-NH3+

(b) 1- chargev) Phosphatidylglycerol

(a) -CH2-CH(OH)-CH2-OH

(b) 1- chargevi) phosphatidylinositol-4,5-bisphosphate

(a) -diphosphopyranose(b) 4- charge

vii) Cardiolipin(a) phosphatidyl-glycerol

(b) -PO4-glycerol with 2 ester-bonded fatty acids

(c) 2- charge4. Galactolipids and Sulfolipids

a) plant membranes use galactose and sulfate instead of phosphateb) phosphate often a limiting nutrient

5. Ether-linked lipidsa) ether bonds are more stable and more resistant to lipasesb) found in archaebacteria

(1) live in extreme environments(2) often as tetraether lipids

(3)

(4) diphytanyl groups twice as long as typical fatty acids in membranesi) one tetraether lipid spans entire thickness of membrane as opposed to halfway in plant and animal cells

6. Sphingolipidsa) Ceramide

(1) -H(2) neutral

b) Sphingomyelin(1) -phosphocholine

(2) -PO3-CH2-CH2-N+(CH3)3(3) 1+ charge

c) Neutral glycolipids(1) glucosylcerebroside

i) found in plasma membraneii) -glucose

d) Lactosylceramide


18

VIII. Lipids


6. Sphingolipids

c) Neutral glycolipids(1) glucosylcerebroside

ii) -glucosed) Lactosylceramide

(1) -Di, tri, or tetrasaccharide(2) -Glu-Gal

e) Ganglioside GM2(1) -complex olicosaccharide(2) contains sialic acid(3) negative charge

f) Glycosphingolipids found as RBC antigens7. Phospholipid and Sphingolipid degradation

a) recycled by lysosome enzymesb) lipases sever ester bonds linking backbone to chainsc) glycosidases cleave sugar units apart

8. Sterolsa) 4 fused carbon rings

(1) 1 - 5C ring(2) 3 - 6C rings

b) Structural lipid form(1) long alkyl chain on C17

c) detergent form(1) charged headgroup(2) amphipathic molecule

d) steroid hormones(1) no long chains(2) no charged groups(3) polar substituents on C17

9. Vitaminsa) Vitamin D

(1)

(2) produced in skin from UV exposure of 7-dehydrocholesterol(3) hydroxylated in kidney and liver to 1,25-dihydroxyvitamin D3

i) regulates calcium ion uptake in intestines(a) deficiency leads to ricketts

b) Vitamin A (retinol)

(1)


19

VIII. Lipids


9. Vitamins

b) Vitamin A (retinol)

(1)

(2) produced form dietary ß-carotenei) made from isoprene units

(a) CH2=C(CH3)-CH=CH2(3) oxidized to cis-retinal

i) visual pigment(a) when exposed to light, becomes trans-retinal

ii) oxidized to retinoic acid(a) hormone

c) Vitamin E

(1)

(2) antioxidanti) reacts with free oxygen radicles to prevent damage to lipids

(3) highly soluble in lipidsd) Vitamin K

(1)

(2) prothrombin activation in blood clottinge) Ubiquinone

(1)

(2) electron transporter in mitochondrial membrane(3) oxidized (quinone) and reduced (quinol) both highly soluble in lipid membrane


20

VIII. Lipids


9. Vitamins

e) Ubiquinone

(2) electron transporter in mitochondrial membrane(3) oxidized (quinone) and reduced (quinol) both highly soluble in lipid membrane

i) can shuttle electrons and protons from one side of membrane to the other

IX. Membranes and TransportA. Lipid aggregates

1. Micellea) spherical structure with hydrophobic interior and hydrophilic exterior

(1) formed from fatty acids and detergentsi) chain has smaller cross-section than head

2. Bilayera) flat double-layer with hydrophilic exterior and hydrophobic core

(1) formed from glycerophospholipids and sphingolipidsi) similar chain and head cross-section

B. Functions of membranes1. physically separate cells from external medium and organelles from cytoplasm

a) important to maintain concentrations of biomoleculesb) segregation of proteins

2. facilitate transport of substrates and ions3. sites of energy conversion in living cells

a) mitochondria, chloroplasts, bacterial plasma membrane4. changes of electrical potential are basis of nervous system5. cell-cell interaction6. contain hormonal and other receptors

C. Cellular membranes1. edges of bilayers are unstable

a) tend to form membrane vesicles2. cellular membranes contain lipids and membrane proteins

a) both freely diffuse in the plane of the membrane3. biomolecular reactions can be more efficient in membrane due to higher probability of collision4. lipid diffusion is slow across membrane

a) diferent composition on each sideb) accelerated by flippase enzymes

5. phase transition temperaturea) in a pure bilayer, below phase transition temperature, lipids are in paracrystalline state, above in fluid state; intermediate, lateral diffusion

but partially orderedb) temperature depends on fatty acid composition of phospholipidsc) in biological membranes (not pure bilayer) uneven phases across membrane

(1) lipid rafts formed by sphingolipids and cholesteroli) ordered lipid surrounded by disordered fluid

ii) stabilized by cholesterol ring interactionsiii) attachment points for peripheral membrane proteins

(a) anchored by covalently-attached acyl chains or glycosylated phosphoinositol derivatives (GPIs)6. Peripheral and integral membrane proteins

a) peripheral(1) attached to membrane by covalently-linked lipid anchors or non-covalent bonds with other proteins

i) GPI anchored proteins are always outside cellii) fatty acyl or prenyl anchors are always inside cell

b) integral(1) immersed in the membrane(2) can be extracted with detergents(3) 2 structural types

i) α-helical bundlesii) ß-barrels

iii) length of transmembrane proteins determined by thickness of bilayer(4) charged residues in proteins mostly outside the membrane or in active site

i) apolar residues inside hydrophobic slab of membraneii) Try and Tyr at head-tail interface


21

IX. Membranes and Transport

C. Cellular membranes

6. Peripheral and integral membrane proteins

b) integral

(4) charged residues in proteins mostly outside the membrane or in active sitei) apolar residues inside hydrophobic slab of membrane

ii) Try and Tyr at head-tail interfaceD. Membrane Fusion

1. Central to many biological processesa) synaptic junctions

(1) vesicle containing neurotransmitters approaches membrane of receptor(2) SNAP and SNARE proteins bind together to draw membranes together(3) void formed and fusion pore allows vesicle contents to enter

E. Membrane diffusion1. diffusion influenced by both concentration and electrical potential gradients

2. ∆μXm+=m∆Ψ–(2.3RT/F)•log([C in]/[Cout])

∆Ψ is normally out–in, inside is negative and outside is positive, so ∆Ψ will also be positive. However when measuring action potentials in neurons, usually in-out is used.

a) ∆μH+=∆Ψ–(60 mV)∆pH(in-out)

For protons m=1 and log [H+in]–log[H+

out] = ∆pH

b) ∆μXm+= –∆G/F

(1) if ∆μXm+ > 0 then since ∆G < 0 diffusion will occur down electrochemical gradient

c) ∆μXm+= electrochemical potential difference

d) F= Faraday constante) ∆Ψ = membrane potentialf) m = charge of particle diffusing

F. Membrane Transporters1. lower activation energy barrier by replacing substrate hydration shell with polar groups along path2. Channels

a) membrane pores that can only transport down electrochemical gradient (passive)b) very high conductance

(1) bind substrate weakly(2) no saturation behaviour

3. Carriersa) rate-limiting step of substrate binding to protein

(1) Michaelis-Menten equation(2) lower rate of transport

b) both active and passive transportc) Example: glucose transporter in erythrocytes

(1) passive carrierplasma glucose concentration higher than within RBCs

(2) V0=(Vmax•[S]out)/(Kt+[S]out)

(3) polar sidechains H-bond with glucose in inside of porei) pore constructed from several helices

4. Cotransporta) two substrates pass through transporter at the same timeb) Symporter

(1) substrates pass through in same directionc) Antiporter

(1) substrates pass through in opposite direction(2) electroneutral ion exchange prevents change in membrane potential(3) electrogenic exchange uses membrane potential to drive active ion transport

5. Types of membrane transport processesa) Active (against gradient)

(1) all active transporters have α-helix domainsi) more flexible than ß-barrel H-bonded structures

(2) Primary active transporti) couple unfavourable transport against ATP hydrolysis

(a) phosphorylation of enzyme drives conformational change(3) Secondary active transport


22

IX. Membranes and Transport

F. Membrane Transporters

5. Types of membrane transport processesa) Active (against gradient)

(2) Primary active transporti) couple unfavourable transport against ATP hydrolysis

(a) phosphorylation of enzyme drives conformational change(3) Secondary active transport

i) couple unfavourable transport with favourable transport of another substance (down gradient)ii) act by "rocker switch" mechanism

(a) substrate binding site exposed alternately to cytoplasm and extracellular space in two states(b) requires that release state has lower substrate binding affinity than first state

b) Passive (along gradient)(1) facillitated diffusion(2) ion channel

i) high conduction ratesii) selectivity

(a) other ions must be excluded

(b) selectivity filter (in K+ channel)i) geometry of pore matches that of hydrated ion

(A) other ions have different hydration sphere geometries(B) electrostatic repulsion between the two ions that can occupy selectivity filter prevent tight binding to channel

(c) aquaporinsi) impermeable to protons

ii) pore reorients water molecules to single fileiii) Asn residues at constriction of pore replace H-bonds, preventing proton hopping (Grotthus mechanism)

iii) can be gated(a) regulated by conformational changes that open and close pore

(b) neuronal Na+ channel is voltage gatedi) opens in response to membrane depolarization

(3) ionophore-mediatedc) simple diffusion

(1) nonpolar(2) down concentration gradient

(3) eg. CO26. ATP synthase

a) reversible

b) converts a proton gradient to ATP if [H+]out > [H+]in

c) if [H+]out << [H+]in, then synthase acts as proton pump(1) must be enough to overcome membrane potential drawing protons inwards

d) protons attracted to negative residue in hydrophobic core of rotor, pressed to other side of membrane as rotor turns7. Intestinal lumen epithelial cells

a) glucose must be pumped from lumen into epithelial cells by Na+-Glc symporter(1) relatively high Glc concentration in epithelial cells

b) pumped into blood by passive uniporter

X. Nucleotides and Nucleic AcidsA. Nucleotides

1. building blocks of nucleic acidsa) linear polymers form nucleic acids

2. Sugarsa) Ribose

(1) aldopentose with OH groups at every carbonb) Deoxyribose

(1) ribose with OH missing at C23. Nitrogenous bases

a) Purines(1) 6 chain and 5 chain double bonded rings fused(2) Adenine

i)


23

X. Nucleotides and Nucleic AcidsA. Nucleotides

3. Nitrogenous basesa) Purines

(2) Adenine

i)

(3) Guanine

i)

b) Pyrimidines(1) single 6 chain doublebonded ring

i) all have at least 1 ketone groupii) Cytosine

(a) 1 ketone, 1 amino

(b)

iii) Thymine(a) found in DNA(b) 2 ketone, 1 methyl

(c)

iv) Uracil(a) replaces thymine in RNA(b) thymine without methy group

(c)

c) attached to sugar's C1 by N-ß-glycosyl bondB. Nucleic Acids

1. successive nucleotides bonded to sugar 3' by phosphodiester bond from phosphate groups on next sugar's 5'a) new nucleotides always added to 3' end

2. shapea) DNA exists as a double helix

(1) Theorized by Watson and Crick(2) proven by Franklin and Wilkins

b) two antiparallel strands complementary to one anotherc) stabilized by stacking interactions

3. compositiona) chargaff rule

(1) A+G=T+C


24

X. Nucleotides and Nucleic Acids

B. Nucleic Acids

3. compositiona) chargaff rule

(1) A+G=T+C(2) purines=pyrimidines

b) A H-bonds with T(1) 2-H bonds

c) G H-bonds with C(1) 3 H-bonds

4. functiona) sequence of nucleotides encodes amino acid sequences of proteins

5. denaturinga) when heated, H-bonds break and strands separateb) when cooled again, helix reforms by annealing

(1) speed depends on whether strands are fully separated6. melting temperature

a) temperature at which 50% of DNA is denaturedb) depends on ratio of CG to AT

(1) CG has 3 H-bonds; increases melting temperature at higher ratios7. Mutations

a) caused by chemical modifications of DNAb) UV exposure

(1) UV radiation causes adjacent thymine bases to bond together into dimers8. RNA

a) mRNA(1) copy of DNA sequence that encodes proteins(2) transfers from chromosome to ribosomes

b) tRNA(1) match amino acids to mRNA

c) rRNA(1) structural component of ribosomes

d) enzymatic RNA(1) process other RNAs

e) Secondary structure(1) variable(2) does not form long double helix

i) short complementary sections form hairpins, bulges and loops(3) non-standard base pairing

i) GU is common

U of S BIOC200 - Biomolecules Final Notes

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