Thermodynamics G= H -T S

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1 Thermodynamics! G= FREE ENERGY (when negative, free energy is released!!)! H= ENTHALPY! S= ENTROPY " higher entropy= higher disorder - NEGATIVE S" LESS disordered (+ G means non spontaneous- absorbed) - POSITIVE S" MORE disordered! Endothermic" H is positive " heat absorbed! Exothermic" H is negative" heat released! Exergonic" G is negative" SPONTANEOUS! Endergonic " G is positive" NON SPONTANEOUS G= H -T S! NOTE: You can NOT tell if a reaction is spontaneous by looking at H" you must consider G!!! STANDARD CONDITIONS - Go - 1 atm - 1M (ph =0) KELVIN (add 273 to get to kelvin from degrees)- BIOCHEM # G o - 1 atm - 298K - 1 M - But well buffered" [H+]" 10^-7 so ph= 7! How do we make an unfavourable reaction favourable?1 1. We want the concentration of products to be smaller than the reactants to endure G is negative" Rate of product removal is faster than product is being made 2. Constant supply of reactant so concentration is higher than product 3. Pair the unfavourable reaction with one that is highly favourable (really big negative G) to make the overall reaction favourable Amino acid dihedral/torsion angles! ω " Angle of rotation around the peptide bond - Max rotation when in trans arrangement" Min rotation when in cis arrangement" 0 - The restriction around the peptide bond is due to resonance - Resulting in a plane, flat arrangement! Φ" PHI" is the bond Ca N!!" PSI" is the bond Ca C! They are not effected by resonance" are single bonds so have free rotation

2 ! Χ" XI" side chains labelled X1 X2 X3" usually staggered arrangement is preferred RAMACHANDRAN PLOT! Plots all phi and psi angels for a single protein! Phi on x axis" psi on y axis! -180"0"+180 scale! Clusters of points" allowed regions! Outside clusters" disallowed regions ALPHA HELX! -57 and -47 BETA SHEET! -130 and +130 How to identify stereochemistry? # ALL natural amino acids are L amino acids # L amino acids were synthesised from L- glyceraldehyde and D amino acids from D- glyceraldehyde # BUT" THIS DOESN T TELL US WHICH WAY IT ROTATES PLANE POLERIZED LIGHT! L means to the left, D to the right BUT this does NOT correspond to L/D amino acids!! Sterocentre" is the ALPHA carbon 1. Look down H-Ca bond 2. Read clockwise 3. If it spells CORN= L amino acids 4. If it does NOT spell CORN= D amino acid AMINO ACIDS! pka" -log10[ka]" tells us the strength of an acid! ph" -log10[h+]" tells us the concentration of an acid! pka = ph " when 50% of the acid has dissociated

3 SPECIAL AMINO ACIDS TO REMEMBER # Histidine! Aromatic ring! But does NOT absorb UV light! Imidazole ring! Pka 10 # Tryptophan! Is aromatic! Absorbs the MOST UV light! Has a indol ring (N in it) # Tyrosine! Aromatic ring! Absorbs the 2 nd most UV light! Although uncharged has a pka of 10! In active site of enzymes can lose H+ to become charged # Phenylalanine! Has an aromatic ring! Absorbs the least UV light out of the 3 # Proline! CIS and TRANS both found (1:4)! Trans still more favourable and common! Restricted PHI angle to -60 # Arganine! Guanadinium group" resonance" positively charged! pka of 12! neutral form barely ever found! What causes proteins to fold? 1. Electrostatic forces a. Point charges + and (discrete) e.g. amino acids such as aspartic acid, glutamic acid, tyrosine, histidine, lysine and arganine - SALT BRIDGES" interaction between + and AND hydrogen bonding b. Dipoles - Transient charge differences - E.g. PEPTIDE BOND" overall the change is neutral" but there is a change separation (- on O and + on N) c. Van der waals - When the atoms are an infinite distance apart" ΔE is 0 - when they get closer together the change in energy becomes more negative (favourable) - until they reach the most stable distance apart - when then get even closer, the change in energy decreases (less negative) - until it reaches a distance where change in energy is 0! This is where it is neither happy/upset being there (same as if they were infinitely separated) - they get so close it becomes really repulsive and the change in energy becomes positive (unfavourable)

4 2. Hydrogen bonding 3. Hydrophobic interactions! Electrostatic forces and hydrogen bonding between polar groups is NOT favoured because it is competing with polar water! Unfolded protein" ordered arrangement of water molecules around the non-polar side chains! This means disorder is LOW (small/negative ΔT" POSITIVE ΔG (not favoured)! When protein is folded" hydrophobic regions in the middle" more free/disordered water molecules" increase in disorder" increase in ΔT" negative ΔG FAVOURED!! THEREFORE: hydrophobic interactions guide protein folding!!!

5 Protein unfolding! When protein is exposed to urea" denature! When urea is removed" re-fold back to native state! Midpoint" is where half the molecules and folded and half are unfolded! Co-operative" meaning it will either be folded or not (not in between) 1. Protein folding is reversible 2. Protein folding is co-operative (2 state system) Christian Alfinsen 1. Treat ribonuclease with urea and beta-mercaptoethanol (β-me) 2. Protein will denature (lose its native state) 3. Dialyze the urea and Beta-ME away 4. Disulphides would re-form due to the presence of a natural oxidant (oxygen) 5. The protein will re-gain function (dialysis and oxidation occur at the same time) BUT in a different experiment 6. He added a chemical oxidant to the unfolded protein first" allowing disulphide bonds to join up RAPIDLY and RANDOMLY 7. THEN he removed the urea and beta-me 8. DIDN T RETURN TO NATIVE STATE! Findings! This experiment shows that disulphide bonds are NOT necessary for proteins folding! The folding occurs first" THEN the disulphide bonds are important for locking in that structure" stabilize protein structure Protein folding in cells! CHAPERONES- stop proteins from misfolding - They bind to the hydrophobic regions of the protein to prevent wrong interaction! Molecular crowding- can prevent accurate folding in cells! Misfolding when it comes off the ribosome- when protein begins to form before the polypeptide has been made CORI CYCLE! Build-up of lactate" acidosis! Lactate in exercising muscles" blood" liver" glucose" blood" muscles" glycogen Energy stores in humans 1. FAT stores" 84% 2. Protein" 15% 3. Glucose/glycogen" 1%

6 KREB S CYCLE SUMMARY Formation of Acetyl CoA" link between Krebs and glycolysis! Pyruvate" Acetyl CoA! Pyruvate dehydrogenase complex (PDC)! NADH! CO2! First control point! + regulated by: pyruvate and ADP! - regulated by : NADH, ATP and Acetyl CoA! Irreversible 1. Acetyl CoA + oxaloacetate" citrate! Citrate synthase! Large negative ΔG! Citrate is highly favoured! Oxaloacetate binds first" conformational change" allows Acetyl CoA to bind! Irreversible 2. Citrate" cis-aconitase" iso-citrate! Aconitase! Large positive ΔG! This means citrate production is preferred BUT due to iso-citrate being used up quickly can drive reaction forward 3. Iso-citrate" a-ketoglutarate! Iso-citrate dehydrogenase! Second control point! NADH! CO2! Irreversible 4. A-ketoglutarate" succinyl CoA! A-ketoglutarate dehydrogenase complex! Third control point! NADH! CO2! Large negative ΔG! Irreversible 5. Succinyl CoA" succinate! Succinyl CoA dehydrogenase! GTP 6. Succinate" fumarate! Succinate synthetase! FADH2 7. Fumarate" carbocation transition state" L-malate! Fumarase 8. L-malate" oxaloacetate! NADH! Large positive ΔG

7 ELECTRON TRANSPORT CHAIN! Albert L. Lehninger" determined that the mitochondria is the site of oxidative phosphorylation in eukaryotes COMPLEX 1! PROTON PUMP! Pumps 4H+ for NADH! **FADH2 SKIPS complex 1**! 42 subunits! Takes NADH from the Citric acid cycle and strips the electrons so NADH + H+" NAD+! Electrons are passed via metal centres to ubiquinone (Q)" causing it to reduce to Ubiquinol (QH2)! Protons are pumped from the matrix to the inter-membrane space COENZYME Q! Lipophilic! Located in the inner mitochondrial membrane! Transfers electrons from complex 1"2"3! Ubiquinone (Q)-oxidised" semi-ubquinone(qh)- only partially reduced" ubiquinol (QH2) COMPLEX 2! NOT A PROTON PUMP! It is an ENZYME!!!! Enzyme succinate dehydrogenase! Converts succinate to fumarate in the citric acid cycle! Converts FADH2" FAD! Electrons are passed via metal centres! Passed to ubiquinone" ubiquinol COMPLEX 3! PROTON PUMP! Pumps 4H+ for NADH! Pumps 4H+ for FADH2! Accepts electrons from ubiquinol! Passes electrons to cytochrome c! 4H+ are pumped out/ NADH CYTOCHROME C! Soluble protein in the inner membrane! It accepts electrons from complex 3" passing it to complex 4! Has a Heme C group" which has a central Fe 3+ atom that is REDUCED to Fe2+ when it accepts electrons from complex 3! Fe2+" Fe3+ when it donates electrons to complex 4 COMPLEX 4! PROTON PUMP

8 Adrenalin signalling! Same downstream effect as glycogen!! Adrenalin binds to its Beta-adrenergic receptor! this activates G protein" adenylate cyclase! increase conversion of ATP to camp! activation of PKA enzyme! quite a slow response" needs to pass from the adrenal glands" blood" liver **our liver is more responsive to glucagon than it is to adrenaline! LIVER! in response to adrenaline! we want an increase in glucose! gluconeogenesis! glycogenolysis MUSCLE! in response to adrenaline! we want to USE glucose! glycolysis! glycogenolysis HOW is this difference achieved? IN BOTH! we want glycogenolysis! so we have the activation of phosphorylase kinase through phosphorylation by PKA! we then have phosphorylation of glycogen phosphorylase! and thus increase in blood glucose DIFFERENCE LIVER! L-pyruvate kinase! This has a serine at position 12! Can be phosphorylated by PKA! and so this can stop glycolysis" increase gluconeogenesis MUSCLE! M-pyruvate kinase! Has an arganine (not a serine)! Can NOT be phosphorylated by PKA! Maintains its function in glycolysis ** muscle glycolysis is NOT inhibited by adrenaline signalling but the liver is**

9 ! Remember they are ENZYMES " which is why we us Km to describe the rate of action! Km is the substrate concentration at which the reaction is at half V max (K m values) 1. GLUT 1" in all mammalian tissues" 1mM! Like GLUT 3" low Km means it is working fast all the time!! We want supply to the brain and tissues to be constant and not dependent on the meal 2. GLUT2" Liver and pancreatic beta cells" 15-20mM! Has a very high Km so, requires high blood glucose levels to work! Only working when we have just eaten 3. GLUT 3" Brain " 1mM 4. GLUT 4" muscle and fat cells" 5mM! Since normal blood glucose is at 5mM this indicates it will always be working at rest! Any rise in glucose levels, will cause up-regulation of GLUT 4! Working at half its maximum at resting glucose levels 5. GLUT 5" Small intestine ALPHA HELIX! CAN exist on their own as they rely on internal hydrogen bonding! 3.6 residues per turn! 5.4 A vertical height per turn! PHI" 57! PSI" 47! Amphiphillic! 3-4 pattern of hydrophobic amino acid side chains - This means for every 7 residues, no. 1 and 4 will be hydrophobic - This means it aligns on one side of the helix - So we can have 2 alpha chains interacting via their hydrophobic sides OR an alpha helix interacting with a bet sheet Determining right from left handed:! Point your thumb at the bottom of the helix! Follow fingers in the direction of the strands! If you travel UP" RIGHT HANDED! If you travel DOWN" LEFT HANDED BETA SHEET! Beta strands cannot exist on their own" rely on inter-chain hydrogen bonding! PHI" -130! PSI" +130! Each residue alternates pointing UP or DOWN the plane" can have one side all hydrophobic and the other all hydrophilic! Can be parallel or anti-parallel (favoured)! Also have a slight twist (Right or left)

10 Beta barrel! Alternating residues are hydrophobic then hydrophilic! Formation of a hydrophobic core and hydrophilic outer surface Beta turns! Irregular secondary structure because phi and psi angles do not repeat!! Their role is to REVERSE the direction of the main chain! Made up of 4 residues! Most common residue" proline " PHI= -60 Type II beta turns NOTE! GLYCINE in position i+2 because it has NO steric hindrance! Allowing it to make a POSITIVE PHI angle needed for the turn! Beta sheets and alpha helices are regular secondary structures as they have repeating phi and psi angles! Beta turns are irregular secondary structures as they have different phi and psi angles (nonrepeating) SUPERSECONDARY STRUCTURE! Alpha-alpha hairpin! Beta-beta hairpin! Beta-alpha-beta TERTIARY STRUCTURE! One polypeptide strand can be folded to have many domains! Domains have core and are made up of 2 or more secondary/super-secondary structures! Each domain has a structure and function! Can have repeating domains" MODULAR NUCLEIC ACIDS! ATP" ribose sugar and adenine base! Nucleotide- base, sugar and phosphate! Nucleoside- base and sugar (NO PHOSPHATE) DNA has a major and minor groove! Major groove" more chemicals exposed in it! Minor groove" no methyl groups exposed! Common chemicals - Methyl groups - Hydrogen atoms