Biochemistry PDF

BIOCHEMISTRY (Section b pages 19-42) Amino acids: 20 natural amino acids primary amino group, carboxyl group, H atom and a R group (central a-carbon) Proline has secondary amine group Enantiomers- all AA except glycine have chiral centre have a d or l configuration —> only L isomer fo...

BIOCHEMISTRY (Section b pages 19-42) Amino acids: 20 natural amino acids primary amino group, carboxyl group, H atom and a R group (central a-carbon) Proline has secondary amine group Enantiomers- all AA except glycine have chiral centre have a d or l configuration —> only L isomer found in proteins D- amino acids can be found in bacteria cell walls+certain antibiotics 20 amino acids: different r groups (side chains)=Different physiochemical properties Glycine- H atom as R group Alanine,valine,leucine,isoleucine, methionine- aliphatic side chains (hydrophobic+ inert) Phenylalanine, tyrosine, tryptophan- aromatic side chain (hydrophobic)  Proline- aliphatic side chain bonded to its amine group = imino acid Cysteine- sulphur containing side chain Arginine, lysine- +ve charged side chain Histidine- basic side chain Aspartic acid, glutamic acid- acidic side chain Asparagine, glutamine, serine, threonine- uncharged, polar, potential to form hydrogen bonds Protein structure: peptide bond via condensation reaction Proteins = sequence of amino acids linked by peptide bonds Peptide bond- covalent bond between the a-amino group of one AA and the a-carboxyl group of another AA Unbranded AA acid chains linked together by peptide bond (25AA)= polypeptide 2x AA linked together via peptide bond= Di peptide Resonance —> free rotation occurs around Bond  primary structure- linear sequence of AA in a specific order sequence determined by sequence of base nucleotides in gene encoding the protein Secondary- folding of regions of polypeptide chain —> a-helix or b-pleated sheet (held by hydrogen bonds) a-helix structure AA acids arrange in regular helix formation Carbonyl oxygen of each peptide bond is H bonded to amino group on $th AA acid away H bonds run parallel to axis of helix Side chains of AA positioned on outside of helix b-pleated sheet structure H bonds form between peptide bonds in different sections of the polypeptide chain Peptide bond = planar—> AA side chains protrude above and below sheet Slightly curved Several b-p sheets —> b-barrel (strong and rigid —> structural proteins) Adjacent pp chains can be parallel or anti parallel due to polarity —> depends on direction they fold Tertiary- 3D arrangement of AA in polypeptide chain ( held by ionic bonds and H bonds and duisulphide bridges if cysteine present) chain folds spontaneously due to electrostatic forces resulting from specific AA side chains Quaternary- multiple polypeptide chains Hydrophobic forces and protein structure; hydrophobic effect- non-polar molecule arranged to minimise contact with water Proteins can arrange 3D structure so nonpolar side chains have minimum contact with aq solvent\ Disulphide bonds- covalent bonds form between s groups in cysteine—> cys residues stabilise 3D structure Haemoglobin + myoglobin oxygen binding proteins Haemoglobin- function- carry O2 in the blood from the lungs to other tissues in the body—> supply cells with O2 required for oxidative phosphorylation Contained in erythrocytes (RBC) Structure- 4 polypeptide chains, 2 alpha , 2 beta , each has haem prosthetic group (quarternary) , can carry 4 oxygen molecules Allosteric - affinity of O2 varies by interactions with other subunits co-operative binding- binding of an O2 to the molecule creates conformational changes that increases the affinity for O2 so more O2 binds Myoglobin: globular protein, single polypeptide chain folded into 8 a helix, haem group located in hydrophobic cleft inside folded polypeptide chain Stores O2 in the tissues Mainly found in skeletal and cardiac muscle No cooperative binding Bohr effect- H+ , co2, (released from carbonic acid —> co2 dissolved in blood), 2,3- biphosphoglycerate promote release of )2 from Haemoglobin —> bind to different parts of the polypeptide chains and prote dissociation of O2 from oxyhemoglobin (< affinity for o2) All act at different sites Fetal Haemoglobin- HbF—> consists of 2 a-chains and 2 y-chains Has higher affinity for o2 —> optimises o2 transfer from maternal to fetal circulation in placenta Nears birth y-chain synthesis is switched off and b-chain synthesis is switched on C1 introduction to enzymes Enzymes catalysts that increase rate of chemical reaction without being changed themselves Highly stereo specific (only certain stereoisomers of a molecule) active site Region that binds to substrate and converts it to product specific 3D T structure and is an open cleft on the surface of enzyme Substrate bound to AS with weak forces —> ES complex substrate specificity determined by properties and spatial arrangement of AA residues forming AS Classification six groups (type of reaction) Each has a unique 4 digit classification number enzyme assays- measures conversion of substrate to product under different conditions coenzymes - transfer groups from one molecule to another (some enzymes require to function) Prosthetic groups- some enzymes require presence to function isoenzymes- differnet forms of enzyme which catalyse the same reaction but exhibit different physical or kinetic properties C2 Thermodynamics Gibbs free energy- if -ve the reaction can occur spontaneously Is +ve the reaction requires input of energy to drive the reaction the energy difference between the substrates energy and activation energy Activation energy must be reached in order for reaction to occur Enzyme lowers activation energy by providing an alternative reaction pathway C3 enzyme kinetics Enzyme activity- expressed by initial rate of reaction being catalysed Substrate concentration: low concentration of substrate = low rate of reaction (less collisions with enzyme= less ESC) Doubling of substrate concentration = doubling of ROR (substrate concentration is directly proportional to rate of reaction) (if hasn’t reached saturation) Too high substrate concentration= fixed reaction rate as a rate is dependant on how fast product can dissociate from enzyme Enzyme concentration: id substrate concentration is saturating then doubling enzyme concentration will double rate Temperature: > temp = > thermal energy of substrate molecules = > proportion of substrate molecules collide with enzyme with sufficient energy to overcome Gibbs free energy of activation = > rate of reaction Too high temp = enzyme 3D tertiary structure denatures as bonds holding the structure vibrate and break = no longer stereospecific = < ROR Ph: optimum pH as pH effects how amino acid functional groups ionise Can also break ionic interactions in 3D structure causing denaturation Michaelis-menten model: enzyme (E)combines with substrate (S)—> enzyme substrate complex (ES)—> products rate constants= k1,k2,k3 Km= Michaelis constant (measure of affinity of an E to its ES) equation describes observations At low (S) the Vo (enzyme velocity/rate) is directly proportional At high (S) the velocity tends towards max Vmax Lineweaver-burk plot: can determine Vmax and Km by measuring Vo at differnet substrate concs—> plotting 1/(s) against 1/Vo (lineweaver-burk plot) y-axis intercept = 1/Vmax X-axis intercept = -1/Km Slope = Km/Vmax (tangent) C4 enzyme inhibition Enzyme inhibition: inhibitor- acts directly on enzyme slowing its catalytic rate Irreversible or reversible Reversible can be a competitive or non-competitive inhibitor (competitive binds to active site whereas non-c binds to allosteric site) Irreversible reaction; often inhibitor forms covalent bond to AA residue at or near active site = permanently inactivates enzyme Ser and Cy’s residues => susceptible as have reactive -OH and -SH groups respectively Reversible competitive inhibition: competitive inhibitor - close structural similarities to normal substrate —> competes with S to bind to AS At high S concentrations the inhibitor will be out-competed in binding to AS Vmax remains constant Km increases as enzymes affinity for substrate decreased slightly Reversible noncompetitive inhibition: non competitive inhibitor- binds on site away from active site—> causes change to 3D tertiary structure —> decrease in catalytic activity Substrate conc can’t overcome noncompetitive inhibition as both can bind at same time Vmax decreases Affinity for substrate of enzyme remains same —> Km unchanged Lecture (cells) Cell= basic unit of life Organelle= small structures within cell with a specific function Cell membrane: function= regulates material entering and exiting cell Structure= phospholipid bilayer, proteins, cholesterol Cytoplasm: function= all cell contents that lie between the membrane and nucleus Cytosol= liquid portion/non-organelles Nucleus: function= regulates DNA and RNA actions Structure= membrane bound, contains DNA, spherical, approx 10% cell volume nuclear envelope- regulates what enters/exits nucleus double layer of lipids/membrane nucleolus- produces RNA (used to make proteins) inside nucleus ( dark region) DNA- info on how to make proteins chromatin- unorganized dna (dark material) Chromosomes- organized dna nucleoplasm amorphous fluid-> contains proteins/RNA/small molecules/ fibrous chromatin Endoplasmic reticulum : highly convoluted single membrane enclosing a single compartment —> netlike mesh work/ tube like channels —> vesicles which bud off ER to transport proteins to Golgi rough ER has ribosomes on outer membrane Protein synthesis + transport Predominant in cells..> protein synthesis smooth ER no ribosomes lipid synthesis + transport Predominant in cells > lipid and drug metabolism functions - detoxification of drugs/ translocation(proteins)/ glycosylation of proteins/ lipid bilayer assembling Ribosomes: protein synthesis rna + protein Vacuole+ vesicles: storage for water, nutrients, waste Membrane bound organelle Lysosomes : vesicles containing digestive enzymes -> digestion/breaking down of worn out cells or organelles / pathogens phagocytosis Nutrients to cell -> lysosomes fuse with food vacuoles -> polymers digested to monomers -> pass to cytosol to enter cell Enzymes work best at pH 5 (organelle creates custom pH) proteins in lysomal membrane pump H+ from cytosol into lysosome —> maintain pH —> prevents leakages of digestive enzymes thus wont digest ourselves  lysosomal storage diseases - metabolic disorders due to lysosomal dysfunction ( result in substrate accumulation in cells) lipids - Gaucher’s disease, niemann-pick disease, Tay sachs Glycogen/ polysaccharides- farmer disease,krabbe disease Proteins- schindlers disease Apoptosis- lysosomes break open and destroy cell Golgi apparatus: structure- 4-6 splattered smooth membrane bound compartment with associated vesicles - Polarized structure functions: protein packaging, modification and transport (make lysosomes ) Mitochondria: oxidative phosphorylation site -> produce ATP from food energy Double membrane bound -> inner membrane folded into crystae—> inside=matrix Large organelles 1nanometer approx  stages of conversion of chemical food energy to ATP energy Oxidative phosphorylation- ETC, ATP synthase, H+ gradient ATP: hydrolysis of ATP releases energy regenerates rapidly mitochondrial disease mutations to mitochondrial DNA occur frequently due to error-prone DNA rep-> when mitochondrion divides the 5-10 DNA copies are divided randomly between 2 mitochondria occurs spontaneously 1 in 4000 children develop by 10 years Cytoskeleton: microfilaments+ micro tubules -> provide structure and support to cell actin filaments anchored underneath plasma membrane = responsible for cell changing shape cytoskeleton and associated motor protiens -> organises + moves cytoplasmic contents microtubules move oraganelles and vesicles Cell movement = polarised and directional Centrioles: microtubules that divide cell during cell division Lecture cell membranes Structure: fluid mosaic model Glycolipids, glycoproteins, protein channels, peripheral membrane proteins integral proteins- embedded and span whole bilayer Peripheral- penetrate one surface of bilayer Membrane proteins Protein channels- facilitated diffusion Protein pumps- active transport Cytoskeleton attachments- support Antigens (proteins and glycoproteins)- cell recognition Tight junctions- protein complexes —> seal neighbouring cells together, regulate selective movement of solutes across epithelium Vesiculation- process that involves formation of vesicles which move substances in and out of cells endocytosis- movement into cell (too large to passively cross membrane) Plasma membrane of cell invaginates> forms pocket around target -> pocket pinches off -> target contained in intracellular vesicle E.g phagocytosis (cellular eating) or pinocytosis (cellular drinking)n Exocytosis- movement out of cell Constitutive (continuous) or regulated by receptors Simple diffusion: phospholipid bilayer is permeable to small gases e.g o2,co2,n2 ,small uncharged polar molecules e.g urea, h2o, ethanol Facilitated diffusion: faster than simple Selective - specific binding sites on transport proteins -> susceptible to competitive+incompetitive competition carrier proteins - bind to specific solute-> undergo conformational change-> transports solute across bilayer -> released -> returns to original shape (slower process) Channel proteins- form water filled pores -> allow ions to pass through Active transport: mediated by carrier proteins coupled to energy source Against conc gradient P class transporters ( ion pumps ) phosphorylation and conformational change occurs to pump ions across membranes ABC transporters DNA composition and structure lecture DNA structure- two polynucleotides arranged into a double helix held by hydrogen bonds between complimentary base pairs (AT) (CG) Bases arranged into triplets called codons -> each codon codes for specific AA Nucleotides Phosphate group, Pentose sugar, nitrogenous base phosphate and sugar form phosphate-sugar backbone adenine=thymine (2 h bonds) Cytosine=-guanine (3 h bonds) A G are purines (two fused rings 6+5 membered ring) C T are pyrimidines (single six membered ring) gene- section of DNA that codes for a specific polypeptide DNA replication mitosis Two strands of dna separated using enzyme-> both strands act as template -> protein brings correct new base pair nucleotide to existing base -> new nucleotides condensed together using enzyme -> forms two new DNA double helix (each with one strand from original DNA and one new strand) DNA condenses-> winds around histone proteins -> further coils _> chromosome Protein synthesis lecture Central dogma= DNA to rna to protein proteins composed of AA 20 AA Transcription: (DNA to mRNA) DNA has genetic code for protein template DNA = too large to leave nuclear pores (double stranded) -> mRNA made leaves nucleus (single stranded) -> acts as template mRNA enters cytoplasm -> binds to ribosome Translation: (mRNA -> polypeptide) decoding of an mRNA message into polypeptide chain mRNA attaches to ribosome at start codon -> tRNA brings anti-codons complimentary to the codon (3 bases) on mRNA -> has specific AA attached How AA bind to cognate tRNA: AA + ATP using enzyme forms amino-acidAMP complex and diphosphate AA-AMP complex + tRNA + calysed by specific tRNA synthase = AA/ tRNA complex and AMP TRNA structure Translation: 1) initiation small subunit complexes with initiation factors on ribosome form base pairs with a special sequence on start codon of mRNA start codon (AUG) on mRNA -> positioned in ribosomal P site -> formyl-methionyl tRNA joins (1st AA) Large subunit on ribosome joins -> GTP hydrolysed to GDP+Pi -> initiation factors leave ribosome 2) elongation 3 binding sites where tRNA can bind -> usually only 2 occupied at a time 3 sites= APE (moves from A->P->E) A site = aminoacyl acceptor site (AA-tRNA in) P site- peptidyl site (bonds form between AA -> holds tRNA with growing AA chain) E site= exit site EFTu catalyzes delivery of AA-tRNA to ribosome (carries AA-tRNA and GTP as a ternary complex to ribosome site-> conformational change when GTP->GDP+Pi -> dissociates) EFG catalyses hydrolysis of GTP->GDP+Pi = conformational change in EFG = change forces tRNAs from A site to P site for next AA-tRNA to join (catalyses translation) peptide bond forms between AA at A and P site-> ribosome moves 1 codon forwards on the mRNA template using EFG-> growing peptide chain now in P site -> A site free for next AA-tRNA-> unloaded tRNA moves to E site -> repeats until stop codon reached 3) termination synthesis continues until stop codon reached Special release factor binds to A site -> polypeptide in P site hydrolysed from tRNA -> leaves via exit tunnel -> tRNA exits ribosome release Catalysed by 2x GTP hydrolysis Ribosomal subunits dissociate-> ready to be reused Antibiotics that work by binding to bacterial ribosomes disrupting their function: streptomycin -> inhibition of initiation Linezolid-> prevention of large subunit binding Tetracycline -> prevention of AA-tRNA binding to A site (common treatment for severe acne) Chloramphenicol -> inhibition of peptide bond formation (topical treatment for bacterial conjunctivitis) erythromycin -> blocking exit tunnel of ribosome (alternative for penicillin due to allergy) macrolide antibiotic amino-glycosides - inhibit initiation e.g streptomycin extremely effective toxic (rarely used) Macrolides- block exit channel of ribosome e.g erythromycin Genome= less complex than proteome single gene can lead to multiple RNA transcripts -> each can be modified to give multiple versions of a single protein Post-translational modification (PTM): modification that occurs to one or more AA on a protein after translation effect protein structure and function Important for regulating cellular activity as proteome changes in response to stimuli E.g phosphorylation Kinases add Pi groups for activation or inactivation Phoshatases hydrolyse Pi group (remove) critical on serine, threonine,tyrosine (have OH group on side chain) residues -> critical for regulation of cellular processes -> cell cycle/ apoptosis/ signal transduction pathways Identification important in heart disease, cancer , neurological disease, diabetes Drugs and central dogma: drugs can bind to DNA directly by covalent adducts or intercalating agents some drugs can inhibit DNA replication Can inhibit transcription and reverse transcription Translation and protein synthesis Cancer lecture 1 What is cancer?; uncontrolled cell division Lifestyle factors and genetics are a major influence Local disease- restricted to tissue of origin Metastasis- cancer cells invade and colonise other cell territories harder to treat All tumours have in common / cancer hallmarks: Leads to an inflammatory microenvironment Causation: Smoking , body weight, physical activity levels, diet (processed/red meat), alcohol , hormones , UV , genetics environmental Uv light, radioactivity, asbestos Viruses Hepatitis-> liver cancer Epstein-Barr -> some forms of lymphoma Papilloma virus -> cervix cancer/ H&N cancer Genetics BRCA1/ BRCA2 in breast cancer How can obesity increase cancer risk in woman Alcohol and cancer : Reproductive hormones/ HRT: Combined oral contraceptives- endometrium, ovary, breast, cervix, liver Oestrogen only HRT- endometrium and ovary Oestrogen-progesterone - breast, endometrium Regular Sunbed use > melanoma risk by 75% before age of 30 Risk factors: moles Fair skinned Bad sunburn esp when child Sun exposure 1) self sufficiency in growth signals normal cells need growth signals to proliferate e.g growth factors, extracellular matrix components, cell-cell adhesion (can’t grow without these) Cancer cells can grow independently Autocrine signalling (cell sectretes hormone/chemical which binds to receptor onsame cell and alters its function) e.g glioblastoma (common form of malignant brain tumour) Alteration in platelet- derived growth factor (pDGF) Cells co-express PDGF + receptor PDGFR Overexpression of message and messenger -> cancer heterotypic signalling- interaction between different cell types Over expression of GF -> default in receptor -> cells can become hyper-responsive to normal levels of GF direct mutation of receptor -> ligand independent signalling (abnormal) -> uncontrolled signalling 2) insensitivity to anti-growth signals the cell cycle : all signals sensed by retinoblastoma protein (Rb) mutation in Rb protien = checkpoints in cell cycle ignored -> cells with mutated dna replicated 3) limitless replication potential karytoptypic disarray- massive cell death pRB (retinoblastoma protein) and p53 = tumour suppressors x pRB or p53 = reduced cell death -> limitless cell replicative potential telomere - tell cell how many divisions it has Abnormal telomere maintenance -> upregulation of telomerase which add 6 base pair repeats onto the ends -> kept above critical threshold = unlimited cell replication 4) evading apoptosis cancer cells to increase -> cell division> cell death Apoptosis= programmed cell death cell is broken down from within by proteins called caspases Extrinsic pathway (initiated outside cell)- usually by t-lymphocytes -> have Fas Ligands on CSM -> bind to Fas receptors on CSM of target cell-> activates intracellular events mediated by FADD (Fas Associated Death domain) -> caspases activate each0ther (caspade cascade)-> initiates apoptosis intrinsic pathway (initiated from within cell)- regulated by maintaining balance between anti-apoptosis proteins ( Bcl-2 and Bcl-x) and pro-apoptotic proteins (Bax + Bak) in the mitochondrial membrane healthy cell pro + anti proteins bind -> blocking action Cell damaged/ not received survival signals -> anti proteins blocked -> pro proteins break channels in mitochondria-> cytochrome C from mitochondria leak into cytoplasm of cell -> binds to Apaf-1 proteins -> complex activates caspase cascade -> apoptosis p53 = tumour suppressor DNA damaged -> identifies and halts cell cycle (G1/S)-> activates DNA repair proteins or apoptosis if damage can’t be repaired absence common in cancer (>50% cases seen mutation or deletion of p53) 5) sustained angiogenesis development of new blood vessels -> carefully regulated in normal tissue Cell must reside within 150-200 micrometers of BV Cancer cells can use angiogenesis to support tumour growth 6) tissue invasion + metastasis metastasis- cancer cells move away from tumour via lymph/blood and colonise dif site (development of secondary tumours) 80-90% cancer deaths cell-adhesion molecules stop invasion and metastasis -> loss-> cancer Lecture cancer 3 Cancer treatments: surgery Radiotherapy Chemotherapy Targeted therapy Surgery not always possible -> dependant on site/ blood supply If localised -> can be fully curative (no metastasis) Not ideal if metastasis occurred-> can be microscopic neoadjuvant- chemo before surgery = shrink tumour before surgery Adjuvant Radiotherapy not always feasible -> depends on site proton beam = latest technology Limits toxicity around tissue can be used alone Used in combination Debunk for surgery Prime immune system for immune targeted anti-cancer drugs Disadvantages Residual cells often more aggressive than pre-treatment Normal tissue damage can increase risk of secondary tumours Chemotherapy targets uncontrolled cell growth ( hallmark of cancer)-> targets cells with rapid cell growth Works in 3 main ways: bind to DNA -> stop DNA replication Inhibit components of DNA replication binds to + disrupts cell components for cell division Bad side effects -> hair loss, sickness, diarrhoea, weakened immune system (WBC and neutrophils depleted) Symptoms of infection-> fever, shivering, breathing rate/heart rate change (Paracetamol reduce fever= may mask symptoms) Personalised therapy using genetic makeup can predict patients ability to metabolise and adverse effects of treatment Uses prognostic markers Targeted therapy secondary protein structure lecture Polypeptide folding is not random: if 50 AA and 3 orientations per residue -> Not all conformations possible due to rigidity of peptide bond Secondary structure bonding Rotation: only certain phi and psi angles form stable conformations Most common = right-handed a-helix and beta strands Hydrogen bond peptide bonds can h bond N acts a H bond donor and O as acceptor A-helix phi -57 and psi -47 angles repeated give right-handed a-helix stabilised by intra-strand h bonds Features (add to other a-helix) Twists clockwise Peptide bonds = trans+ planar One complete turn every 3.6 AA  amphipathic helix- has hydrophilic + hydrophobic forces Hydrophilic AA on one side and hydrophobic residues on other Can be used to associate protein to a membrane Membrane association: - Complex helices: a-helices can wind around each other -> form ‘coiled coils’ = extremely stable / stiff/ supportive Found in fibrous structural proteins e.g keratin, myosin Ferritin: iron storage protein -> 4500 Fe3+ enclosed (reduces toxicity) Intracellular cytosolic protein Globular protein- water sol + 24 subunits with helices bundles reduces toxicity + maintains solubility Important in iron deficiency and overload Transferrin: iron binds to in blood plasma -> transport to tissues with transferrin receptor 1 e.g erythroblasts to bone marrow Binds to 2 Fe3+ binding site has AA with electronegative and bidentate carbonate hydrogen bonded to arginine side chain and N terminus Ferritin and transferrin in as diagnostic markers iron deficiency anaemia(IDA) Serum iron test shows low iron levels (alone not suffice to diagnose) serum ferritin test will show lower levels Serum transferrin will show higher levels of protein (total iron binding capacity test will reflect lower transferrin saturation of iron) Performing all tests increases chance of correct diagnosis A-keratin: present in hair + wool Two helices wrapped together -> coiled coil -> disulphide bonds + non-covalent interactions Rigid + strong + insoluble A-helix breakers glycine + proline mainly found in bends Glycine= flexible -> easy rotation -> loses rigidity Proline= cyclic imino acid -> often serve as a-helix breakers-> often at turns or boundaries Beta sheets: (add to other) parallel beta sheet = peptide in extended form ( 5 or more residue strands run in same direction (N-C terminal) anti parallel beta-sheet Stands alternately up and down Strands run in opposite direction E.g fibroin (silk) b-pleated sheet side chains alternately up and down A-carbon tetrahedral and peptide bond planar = extended structure appears pleated R groups lie perpendicular Multiple provide toughness +rigidity to structure Turns and bends in structure change in direction in proteins structure = b-turn used (type 1 or 2) Often proline or glycine proline has fixed phi angle -> bend Glycine = very small side chain + flexible -> fits in turns misturns in proteins can cause nuerogenative disorders tertiary protein structure lecture Loops+ bends controls size + shape Loops- contain stretches of hydrophilic residues (found on surfaces of proteins) Used to connect a-helix and b-sheets Turns- same as loop but less than 5 residues e.g b turns structure complexity: motifs- arrangement of 2 > secondary structures e.g b-a-b (2 parallel beta strands connected by alpha helix) Domain- many motifs folded-> stable self-contained tertiary s unit Subunit- combines domains tertiary structure- subunit Quaternary- multiple subunits Homomultimetric proteins- identicle subunits Heteromultimetric protein- dif subunits Hydrophobic bonding: stable if G is negative -> no energy in required High entropy is desired water has solvent = weak h-bond network (dynamically exchanges)-> h2o molecules line up around hydrophobic solute (clusters together to present minimum surface area with water) to try to maintain total h bond energy-> forms solvation shell -> increase entropy Ionic/elecrostatic forces: Stop oxidation of Fe2+ in heam proteins Fe2+ oxidised to Fe3+ when o2 binds to haem in solution (o2 forms superoxide) protein prevents this -> hosts the haem/ stops oxidation / provides pocket for oxygen binding Quarternary structure of proteins lecture Haemoglobin pull on helix F = movement of corners of structure helix Movement transmits alpha-beta interface over next haem -> eases movement of next Fe into haem plane which assists next o2 to bind (cooperative binding) The principal protein involved is haemoglobin. Haemoglobin is a quaternary protein composed of 4 polypeptide chains: two beta chains and two alpha chains (each identified as a subunit). All 4 subunits contain a single heme group. The ferrous iron (Fe2+) in each haem group is enclosed in a planar organic porphyrin ring and each iron molecule is attached to a histidine residue. Deoxyhaemoglobin is haemoglobin with the haem groups in their T-state which is unstable( due to pocket of positive charge at centre of tetramere) Oxygen binds to Fe2+ in deoxyhemoglobin which results in a conformational change. Fe2+ is pulled above the plane also pulling the histidine residue towards itself. The corners of the quaternary protein structure change shape and the helix movement pulls the alpha-beta interface over the next heme. This eases the movement of the next Fe2+ into the haem plane allowing the next o2 to bind. When oxygen is bound to the haem group oxyhaemoglobin is formed and the haem is in its more stable R-state. This process allows cooperative binding as the haemoglobin's affinity for oxygen increases as more oxygen molecules bind forming more stable R-state haem conformations. Allosterity and 2,3 biphosphoglyceric acid BPG binds to Haemoglobin central cavity of subunits (only present in deoxygenated Hb/ T state) -> stabilises T-state which weakens o2 binding / released when o2 binds Foetal Hb x bind In Fb Hb histidine residue is replaced by Ser enzymes lecture Enzymes overview: Enzymes: (add to other notes) proteins -> can be RNA molecules (ribozymes) some need cofactors / coenzymes e.g ca2+, Na+ or ATP/NADH 6 classes Oxidoreductases (oxidation+reduction) Hydrolases (hydrolysis reactions addition of h2o) Lysases (addition or removal of groups to form double bond) Isomerases (isomerization/ intramolecular group transfer) Ligands ( ligation / forming bond of two substrates using energy from atp hydrolysis) k>1 -> product favoured K=1 -> no preference K reactants favoured When G transition state Usually too unstable (>energy) -> transient= short lived -> either goes back to substrate or forward to product Gibbs energy determines which is more favourable enzyme bound transition state = more stable ( rate independent of reactant conc -> V= k 1st order -> rate varies with conc of single reactant -> V= k[A] 2nd order -> rate varies with two reactants -> V=k[A][B] Assumptions of Km (add to previous notes) production of product remains linear Conc of substrate > enzyme conc Single enzyme forms product Negligible product without enzyme No cooperativity, allosteric effects or inhibition Km constant conc of substrate required to reach half maximal velocity Half AS are full Not a equilibrium constant Doesn’t give idea of strength of binding to substrate unless k-1>> Kcat High Km = weak binding Kcat = turn over number / no of sub molecules processed per s per active site when enzyme is fully saturated with substrate (Vmax) 1) cyclooxygenase(COX) converts arachidonic acid->prostagladins (NSAID drug) 2)transpeptidase -> necessary for cross linking of bacterial walls 3)beta-lactamases produced by bacteria ->prevents them inactivating certain beta-lactam antibiotics 4) dihydrofolate reductase (DHFR) -> converts dihydrofolate-> tetrahydrofolate ->THF necessary for synthesis of nucleotides for DNA/RNA -> prevents cell division 5) reverse transcriptase -> HIV infected cells use to make new viruses ( replicate their genetic material) Drugs diseases and enzyme inhibitors Enzymes can be used as : diagnostics Therapeutics Modes of inhibition Drug target Hormones derived from tyrosine: AA tyrosine hydroxylase (TH) deficiency Rare metabolic disorder Reduces L-DOPA + dopamine needed for motor control and movement causes Parkinson’s disease- caused by deficiency in dopamine -> treatment (carbidopa/ levodopa)  dopamine beta-hydroxylase deficiency causes deficiency of norepinephrine (noradrenaline) ans epinephrine (adrenaline) fight or flight response-> Adrenaline- raises heart rate, breathing , physical strength noradrenaline- maintain bp+ energy levels , regulates mood Deficiency symptoms-> dizzy, lack of coordination treatment -> droxidopa (prodrug of norepinephrine/ noradrenaline) Phenylketonuria genetic disease -> mutation in PAH gene Accumulation of toxic levels of phenylalanine due to lack of enzyme phenylalanine hydroxylase (not broken down) Phenylalanine found in protein containing foods and some sweeteners Penicillin and aspirin irreversible inhibitors: Penicillin : Transpeptidase- catalyzes the nucleophilic carbonyl substitution required for the cross-linking of peptidoglycan in bacterial cell walls.-  aspirin methotrexate and AZT - reversible inhibitors methotrexate methatrexate inhibits enzyme dihydrofolate reductase (DHFR) which converts DHFR to tetrahydrofolate (THF) THF- a co-enzyme necessary for nucleotide purine+ pyrimidine synthesis methatrexate binds 100x more strongly than folate Used as cancer treatment AZT - azidothymidine (zidovudine) inhibits reverse transcriptase needed for HIV replication Thymidine analog (replace OH group with azide group) Prevents DNA polymerase condensing nucleotides HIV treatment Interactions between molecules lecture Wallace’s rule %C-G and %A-T base pair content in DNA predicts temp at with DNA helix speparates strands (Tm) adding C-G pair will increase Tm by 4 degrees and A-T by 2 Applies to short segments of up to 20 base pairs Packing interactions aromatic rings can pack together -> pie-pie stacking Occurs for amino acids Phe, Try, Trp, nucleic bases, some ligands Penicillin G serine residue on enzyme transpeptidase attacks beta-lactate ring and acylates enzyme -> allows structural cross-linking in bacterial cell wall penicillin acts as inhibitor (has serine residue) Oregano-phosphate chemical weapons and insecticides for nerve impulse acetylcholine hydrolysed by acetylcholinesterase Enzyme has a serine residue -> allows catalytic activity oregano-phosphate insecticides (e.g parathion/insecticide or sarin/ chemical weapon) form covalent bond with serine group on acetylcholinesterase -> inhibition -> paralysis +death intro to pharmacogenomics lecture study of how single gene can impact interactions between one specific medicine or a group and the body Genome- all genes + DNA between those genes Exome- genes that code for proteins non-coding and coding regions in DNA Genetic variation types 1) deletion delete all or part of gene May cause frame shift in RNA copy sequence 2) copy number variants if genes that code for specific enzymes are duplicated -> result in fast metabolism of drugs -> can result in toxicity 3) chromosomal rearrangement translocation Genetic rearrangement between dif chromosomes or within same chromosome 4) single nucleotide polymorphism One base is substituted or a single base is inserted or deleted may cause a frameshift or premature a stop codon Frameshifts in translation -> ribosome reads genetic code at start codon -> reads 3 nucleotides at a time (codon)-> spec for a AA Inserting or deleting a base can result in sequence change and code for different AA sequence Premature stop codons base change may convert codon for AA into a stop codon Whole protein isn’t translated in translation Concepts of health and disease lecture Health- state of complete mental and social well being Disease- presence of some pathology or abnormality in a part of the body Illness- experience of loss of health Health measured by`: mortality - life expectancy at birth Morbidity (consultation rates, hospital referrals, disease registers) Quality adjusted life years Illness/ health is objective Disease is objective (have it or don’t) How society deals with illness; 1) social norms (dependant on culture) 2)deviance (non-conformity to societal norms)