LSM2106 Exam Lecture Notes PDF

LSM2106 Lecture 1 - Cell and Water Self learning Roadmap 1 - cell and macromolecules 1. Learn the structure and function of organelles in the cell 2. Learn the four macromolecules 3. solutes, solvents, solutions a. Types of sol...

LSM2106 Lecture 1 - Cell and Water Self learning Roadmap 1 - cell and macromolecules 1. Learn the structure and function of organelles in the cell 2. Learn the four macromolecules 3. solutes, solvents, solutions a. Types of solutes i. Ionic 1. hydrophilic ii. Polar 1. Hydrophilic 2. Forms HB with water iii. Non-polar 1. Hydrophobic 2. Attracted to each other in water 4. Types of bonds 5. Hydrophilic. hydrophobic, amphipathic 6. Water a. Covalent > ionic > hydrogen > van der waals b. can form HB is a weak bond that can break easily (in this context) c. proton cannot exist in aqueous solution due to its positive charge, H3O+ Lecture 2 - pH pH - logarithmic measure of H+ conc in a solution ⇒ pH = -log [H+] Low pH = more H+ LSM2106 1  High pH = less H+ many biochemical reactions only occur at certain specific pH levels [H+] can vary greatly, and it would be more straightforward to communicate in pH than [H+] pH measurement probe litmus paper [H+] in water 10^-7M in water pH = 7 water in in equilibrium → pOH of water also 7 pOH + pH =14 decrease in pH by 1 unit → 10x increase in [H+] How is [H+] increased in a solution acid dissociates to produce [H+] strong acids dissociates completely into ions weak acids dissociates partially **mostly dealing with these Acid increases [H+] and decrease pH How is [H+] decreased in a solution bases accepts H+ decreases [H+] and increases pH Water autoionisation of water acts as both acid and base Acid-base pairs acids ↔ conjugate base (nomenclature: -ate) base ↔ conjugate acid (nomenclature: -ic) Bronsted -Lowry definition LSM2106 2  Acids = proton donors Bases = proton acceptors Acid & bases low Ka → high pKa → weak acid * high Ka → low pKa → strong acid * Ka tells you the acid strength (increasing Ka → increasing strength of acid ← decreasing pKa) Ka 1 = strong acid Henderson-Hasselback eqn** pH = pKa + log [base] / [acid] if base > acid → pH > pKa = [base] predominates at high pH if acid > base → pH< pKa = [acid] predominates at low pH drugs are weak acids or weak bases because uncharged molecules pass through membranes more easily/ readily Acid form predominates at low pH - it is uncharged Buffer - ability to resist changes in pH when an acid or base is added to it Removes H+ from the solution when they are in excess Donates H+ to the solution when they are low Typically weak acids where [HA] approx equal to [A-] When excess H+ added, combines with A- When excess OH- added, neutralises H+ Buffering capacity of amino acids will influence the capacity of certain proteins to act as buffers certain amino acids can protonate and deprotonate Do not use Henderson-Hasselbalch for cases where [A-] or [HA] ≈ 0 Polyprotic acids = dissociates multiple H+ LSM2106 3  multiple Ka values can buffer in multiple ranges Importance of [H+] for living things a small change in pH can amount to a large difference in [H+] regulation of pH in the blood follows a very narrow pH range in the blood, the [H+] is regulated by a buffer system known as the CO2 - Bicarbonate buffer system [H2CO3] regulated by breathing and the amt of CO2 in the blood → regulate blood pH kidney also regulate [HCO3-] do it but its slower compared to the lungs Other buffering systems phosphate system bicarbonate system amino acids and proteins Lecture 3 - Amino Acids Animo acids major constituents for brains, nerve, muscle, blood, skin and internal organs 3 different ways to identify/ name animo acids Why is the 1 letter abbreviation needed data bases of sequences will be easier to be record and takes less space bioinformatics mainly uses the 1 letter abbr 20 amino acids LSM2106 4  amino acids come from our diet = diverse diet is impt to provide the body with a variety of amino acids amino acids can be manufactured but a source is needed → come from glucose, which also comes form our diet Structure of amino acid: amino group + a - carbon atom + carboxyl group (same for all amino acids) R group/ side chain differentiates the different types of amino acids a - carbon of amino acids are chiral centres (except glycine) = only L isomers of amino acids exist in proteins the biosynthetic pathways that produce amino acids are stereospecific, and most generate only L-isomers Classification of amino acids grouped according to the characteristics of their R groups grouping by properties polar vs non polar charged vs uncharged (subgroups of polar side groups) Neutral (uncharged) LSM2106 5  Acidic (charged) - contain carboxylic acid Basic (charged) - amino groups list of amino acids will be provided for CA1 and final exam Ionisable groups in amino acids + protonation. deprotonation of amino groups amino acids are weak acids all amino acids have at least 2 acid - base group (a-carbonyl and a- amino grp) amino acids with ionisable side chains have more At pH2, carboxylic acid is deprotonated At pH9, amino group is deprotonated At pH = 7, the amino group will be in the acid form while the carboxylic group will be in its basic form pI = (pKa1 + pKa2) / 2 Isoelectric point pH at which the amino acid has a neutral charge - both positive and negative are equal and charges “cancel out” zwitterions ⇒ net charge = 0 At low pH, [H+] is high, amino group is protonated and the net charge is positive amino group has pKa ~ 9 At high pH [OH-] is high, carboxylic group is deprotonated and the net charge is negative LSM2106 6  carboxylic group has pKa ~2 Finding the point by taking the average of pKa values of amino group and carboxylic groups ( and side chain if the side chain is charged) Acid/ base properties of amino acids amphoteric and neutral pH table of pKa values will be provided the charge of an amino acid is determined by the pKa of each group that has dissociable protons some amino acids will have 3 pKa values because of their side chains All amino acids are polyprotic acids Histidine LSM2106 7  Non standard amino acids - produced from modifications of standard amino acids modified after incorporation into a protein Post-translational modification (modification to amino acids after protein has been synthesized by a ribosome) specificity with regard to: protein residue modification under what condition diverse functions - many regulate protein activity Methods: acetylation many proteins are acetylated at the terminal amine group to prevent degradation by our cells nitrogen is modified by adding an acetyl (-C=OCH3) group hydroxylation of proline hydroxyproline has a hydrogen bonding side chain seen in collagen, the most abundant protein in our body, with proline being the most abundant amino acid in collagen. collagen modifies the structure of proline by adding a hydroxyl group to produce hydroxyproline to increase stability Hydroxylation can takeplace on several amino acids, like lysine, asparagine, aspartate and histidine, BUT The most frequently hydroxylated amino acid residue in human proteins is PROLINE phosphorylation of serine/ threonin or tyrosine used in cell signalling -OH group of Tyr, Ser, Thr can be replaced by PO4H2 Phosphate group is negatively charged at neutral pH LSM2106 8  acts as a switch to activate/ deactivate phosphorylation by kinase, dephosphorylation by phosphatase phosphorylation requires ATP kinase phosphorylation cascade Cysteine forms disulfide linkages bonds 2 cysteine side chains together, links 2 amino acid chains or 2 parts of the same chain affects how the protein will fold - keeps amino acid chains tgt disulfide bonds are more common in proteins outside of cells, where the environment is more oxidising disulfide linkages form after proteins are folded and stabilise folded structure, or keep multiple amino acid chains together (in some proteins) Cleavage of polypeptides proteins synthesised in the inactive form e.g. digestive enzymes, fibrin cleave to activate biologically active amino acids that do not incorporate into a protein/ not found in proteins (e.g. hormones or neurotransmitters) Specific biological function GABA (neurotransmitter) synthesised from glutamate Dopamine (neurotransmitter) synthesised from tyrosine Taurine (essential for development) formed by the decarboxylation of cysteine Histamine (neurotransmitter) Thyroxine (hormone) LSM2106 9  Epinephrine (neurotransmitter and hormone) Metabolic precursors to important molecules Tryptophan largest amino acid precursor to several compounds that affect the brain (e.g. serotonin, melatonin) may cause sleepiness Tyrosine Precursors to neurotransmitters and hormones (e.g. dopamine, epinephrine, adrenaline) selenocysteine (exception: non-standard amino acid that can be incorporated into the protein) 21st amino acid selenocysteine has a structure similar to cysteine but with an atom of selenium taking the place of the usual sulfur Under some specific conditions, the UGA codon (stop codon) is used to incorporate selenocysteine into proteins selenocysteine is added in stead of the stop codon, so translation continues UGA codon (usually a stop codon) is used to incorporate selenocysteine into proteins Amino acids not found in proteins some are metabolic precursors some have specific biological function Lecture 4 - Proteins SARS CoV 2 binds to the receptor that is expressed on our lung cells What is a protein Central dogma of biology = DNA → RNA → protein Protein is a linear polymer of amino acids LSM2106 10  1st amino acid is always methionine - however methionine is often removed from the protein Although Methionine is the first amino acid incorporated into any new protein, it is not always the first amino acid in mature proteins - in many protiens, methionine is removed after translation Condensation/ dehydration reaction to form the peptide bond In the peptide bond, the carboxy and amino groups are no longer able to undergo protonation/ deprotonation as these groups are not covalently bonded Amino terminus (N terminus, amino acid 1) and carboxy terminus (C) left to right → amino acids sequences are directional (same direction as protein synthesis, from N to C terminus) ends are ionisable Constant backbone Variable side chains Peptide (shorter) vs protein/ polypeptide (longer, >50 a.a.) Dipeptide (2), tripeptide (3), oligopeptide (12-20) aka residues Properties of protiens Molecular weight of a protein expressed in dalton mean molecular weight of an amino acid residue is about 110 dalton SDS-PAGE = a technique for separating mixtures of different size, similar to gel electrophoresis Isoelectric point = the pH at which the molecule carries no net charge pH < pI, then protein charge + pH > pI, then protein charge - Applications of proteins diagnostics based on the charge of the proteins at a given pH, we can use this information of the profile LSM2106 11  Important for allowing the protein to migrate from cathode to anode When a cell is infected, some proteins may be expressed more, changing the profile When cell dies, protein is released, which is why there is more CK in serum Structure of polypeptide chains Proteins spontaneously form their native conformation unfolded → intermediate (shifts between different possibilities of conformation until it settles onto the most energetically favourable state) → folded The properties of the side chain will determine the native conformation (in water) weak interactions hydrophobic interactions hydrogen bond ionic bond van der waals force covalent bonding disulfide bridges Factors which disrupt these (weak noncovalent) interactions cause proteins to denature (unfold) heat organic solvent urea low and high pH detergent guanidine hydrochloride Primary structure is the linear sequence of amino acids as encoded by DNA LSM2106 12  So long as the properties of the amino acids side chains are the same, the protein formed can still be the same (cow insulin can be used for humans even though the sequence is not exactly the same - nothing much will happen if you switch the isoleucine with valine) BUT switch glutamic acid with valine → sickle cell anemia → shape of the protein will be very different and the red blood cells with aggregate and become sickle shaped Amino acid substitution in proteins from different species Conservative = substitution of an amino acid by another amino acid of similar polarity or charge Non conservative = substitution involving replacement of an amino acid by another of different polarity or charge Invariant residues = amino acid found at the same position in different species (critical for the structure or function of the protein, cannot be changed) Secondary structure does not involve the side chain of the amino acids alpha helix - right-handed coil stabilised by HB between the amine and carboxyl groups of nearby amino acids Beta sheet - HB stabilise two or more adjacent strands of amino acids parallel antiparallel Tertiary structure 3D structure shape determined by the characteristics of the amino acids & R- group interactions Type of interactions: HB, ionic, hydrophobic, disuphide many proteins form globular shapes with hydrophobic side chains sheltered inside facing away from the aqueous environment while hydrophilic R groups are on the outside of the molecule can form HB with water LSM2106 13  membrane bound proteins have hydrophobic residues clustered together on the outside, so that they can interact with he lipids in the membrane wide variety of tertiary structures as there is a large variation in protein sizes and amino acid sequences Protein starts at amino acid 1 - NH2 group Quaternary structure not all have quaternary structure Multiple polypeptide chains Subunits are bonded by non-covalent interactions = protein complex each subunit is a separate amino acid chain advantages: oligomers are more stable than dissociated subunits (prolong the life of proteins in vivo) active sites can be formed by residues from adjacent subunits/ chains error of synthesis is greater for longer polypeptide chains (so it might be better to just split the polypeptide into smaller segments before assembling the whole thing, which will be less prone to errors) subunit interactions: allosteric effects Domain Part of the protein that folds independently into compact, stable (globular) structures a domain is the modular unit from which many larger proteins are constructed each domain can be associated with a particular function Kinase - a protein that adds a phosphate to another molecule E.g. RTKs - different receptors with similar kinase domain but different ligand binding domains Linked to other domains covalently (in the same polypeptide chain) LSM2106 14  NOT the same as a subunit Protein folding protein misfolding can lead to diseases end up with wrong function/ dysfunctional Protein misfolding occurs regularly there are ways to control the quality of the proteins producted heat-shock proteins (hsp) to degrade wrongly folded proteins At the molten globular stage Can the folding be corrected or not? If not, the protein is trashed, but if it can be corrected, the proteins will be refolded Help to refold misfolded proteins that are still “save-abe” Molecular chaperones help proteins fold properly molecular chaperones are proteins which assist other proteins to fold correctly in cells can prevent or reverse midfolding examples are heat shock proteins (hsp) Hsp-60 = a chaperonin that captures and acts on fully synthesised proteins, allows the protein to fold in a more favourable environment Hsp-70 = acts early on in the folding, aided by hsp40 and many cycles of ATP hydrolysis are required to fold a polypeptide chain properly Proteosome degrades misfolded proteins Neurodegenerative diseases Prion - diseases protein will cause normal protein to misfold into diseased protein when a healthy protein templates onto another protein is can lead to the misfolding of the prion protein, which leads to aggregation all it takes is for the interaction between a healthy protein and a diseased protein to convert the healthy protein into a diseased LSM2106 15  protein normal form of the protein is normally present in the brain and consists of a helix when the protein induces another prion protein to misfold, then there will be both a helix and b sheet insensitive to protease forms insoluble fibres → cell death Mad cow disease How to determine the primary structure of proteins sequence of aa original method: direct sequencing now, the main approach is prediction from DNA or RNA sequences mass spec x-ray crystallography NMR Proteins function Proteins as buffers - due to ionisable side chains (possess basic (lysine, arginine, histidine) and acidic groups(glutamate, aspartate)) proteins buffers are mainly intracellular haemoglobin as part of the buffering system of the blood protein molecules possess basic and acidic groups which act as H+ acceptors or donors respectively Histidine as a buffer - side chain pKa is around 6 close to blood pH of around 7.4, terminal amino group pKa ~8 can also buffer at neutral pH, only histidine residue (pKa~6.0) in proteins act as a buffer component Biologically active oligopeptides small peptides that can be synthesised in the lab oxytocin (neurotransmitter) LSM2106 16  glucagon (hormone) Dipeptides aspartame - nutrasweet Pentapeptides leucine enkephalin methionine enkephalin Cyclic peptides similar in structure (only 1 amino acid difference) but very different in function oxytocin vs vasopresin Lecture 5 - forms and functions of Enzymes Enzyme protein catalyst that increases the rate of a reaction not consumed - returned back to normal exception: ribozyme - RNA acts like the enzyme, catalyses the cleavage and synthesis of phosphodiester bonds active site 3D conformation usually forming a cleft binding by weak forces - quick binding and unbinding Classifications : name -ase Major classes 1. Oxidoreductase - redox 2. Transferases - transfer of functional groups 3. Hydrolases - hydrolysis 4. Lyases - group elimination to form double bonds 5. Isomerases - isomerisation 6. Ligases - bond formation coupled with ATP hydrolysis Features of enzyme: LSM2106 17  1. Higher reaction rates 2. Milder reaction conditions (compared to chemical catalysts) 3. Capacity for regulation (Lect 7) 4. Greater reaction specificity a. without the enzyme the phosphate group can technically react to the other hydroxyl groups b. active site provides the unique combination - the specificity controlled by 3D structure of enzyme Cofactors some enzymes are associated with non-protein components which are required for enzymatic activity inorganic metal ions e.g. Mg2+ ion for hexokinase, Zn2+ ion for carbonic anhydrase and alcohol dehydrogenase coenzymes (organic molecules) vitamins are coenzyme precursors e.g. some enzymes are inactive without their coenzymes NAD+ is the coenzyme (reduced) in the reaction of ethanol with ADH (as enzyme) and NAD+ into acetaldehyde although NADH is produced, it will be oxidised into NAD+ in a separate reaction, thus it is not consumed alcohol consumption stuff Example: alcohol dehydrogenase ethanol + NAD+ (oxidised form) →ADH→ acetaldehyde + NADH (reduced form) + H+ ethanol is the only substrate ADH is alcohol dehydrogenase (enzyme) NAD+ serves as its coenzyme nicotinamide adenine dinucleotide NAD+ oxidises ethanol LSM2106 18  ethanol is oxidised to acetaldehyde whereas NAD+ is reduced to NADH NADH will be reoxidised to NAD+ in an independent reaction for reuse → not consumed in the reaction alcohol group becomes aldehyde group aldehyde dehydrogenase (ALDH) converts (toxic) acetaldehyde into acetic acid mutation in ALDH2 reduces its activity → takes longer time to convert acetaldehyde into acetic acid → “alcohol flush” Activation energy and transition state diagram X++ is the transition state - the point of highest free energy delta G++ is the activation energy or Ea when equilibrium favours products, Keq will be large, lnKeq will be positive, deltaG = -RTlnKeq a’) since inhibitor binds more strongly to free E than ES, competitive effect is stronger, Km will have a net increase dissociation constant for free E will be smaller since inhibitor binds more strongly to free enzyme → Ki smaller than Ki’ → a larger than a’ → a/a’ > 1, Km increase How does the noncompetitive inhibitor inhibit the binding and catalytic steps Summary table Vmax Km Type of inhibition LSM2106 30  Enzyme 100 5 Enzyme + inhibitor A 20 1 uncompetitive Enzyme + inhibitor B 100 20 competitive Enzyme + inhibitor C 10 10 mixed noncompetitive Enzyme + inhibitor D 20 5 pure noncompetitive Lecture 7 - Enzyme regulation control of enzyme synthesis amounts control of enzyme activity 1. Subcellular localisation Hexokinase in charge of first step of glycolysis, phosphorylation of glucose to glucose-6-phosphate using ATP once glucose has been phosphorylated by hexokinase into G6P, it is no longer able to pass through the cell membrane, effectively trapping it within the cell and allowing the Krebs cycle to continue hexokinase has 4 isoenzymes (isoenzymes catalyse the same chemical reaction but are encoded by different genes, hence might be expressed at different tissues and exhibit different regulatory properties): Hexokinase I, II, III present in most tissues except liver; broad substrate specificity Hexokinase IV (aka Glucokinase) predominates in the liver (where glucose concentration is much higher) Liver Brain Fasting: produce and release Does not produce glucose glucose Fed condition: take up and store Utilises glucose as major fuel under fed or glucose fasting conditions Expresses glucokinase Predominantly expresses hexokinase I LSM2106 31  when glucose concentration is low hexokinase predominant in the brain hexokinase activity is much higher than glucokinase in the liver limited glucose in bloodstream brain is able to fully utilise the glucose brain more impt than liver liver will just maintain minimal level to prevent liver from competing with brain for glucose at higher glucose concentration, glucokinase activity is much higher than hexokinase a lot of glucose in bloodstream as glucose concentration increases, glucokinase activity also increases effective in remove the surge of glucose after feeding to prevent high blood sugar LSM2106 32  Sub cellular localisation of glucokinase (GK) in hepatocyte (liver cells) In order for glucokinase to function it has to be present in the cytosome Glucokinase regulatory protein (GKRP) can associate with GK to form a complex that is predominantly present in the nucleus (inactive form) glucose promotes dissociation (activation) fructose 6 phosphate promotes association (inactivation) When blood glucose drops don’t want hepatocyte to compete with the brain want the glucokinase activity reduced glucokinase can be suppressed in the nucleus to reduce the amount of glucose catalysed into G6P no glucose, so f6p is relatively higher in amount f6p induced GKRP to bind and sequester GK in the nucleus, where it is segregated from glycolysis overall promotion of association of complex hence activity of glucokinase reduced After a meal, surge of glucose hepatocyte want to quickly utilise glucose to reduce high blood sugar High glucose over f6p LSM2106 33  overall promoting dissociation GK activity increase ways to regulate GK activity Enzyme’s own kinetics property (high Km) Regulation of enzyme activity: localisation (GK-GKRP in nucleus) Regulation of enzyme synthesis amounts: transcriptional regulation by insulin when blood glucose level rises, B-cells of pancrease increase insulin release, about half of the newly secreted insulin is extracted by the liver insulin promotes transcription of the glucokinase gene, resulting in an increase in glucokinase synthesis when blood glucose level rises alteration in enzyme levels as a result of induction or repression of protein synthesis are slow 2. Proteolytic cleavage Zymogens (Proenzymes) are inactive enzyme precursor that require cleavage to be activated a. Proteolytic enzymes of the digestive tract after a meal, the proteins will enter the digestive tract proteases will cut up/ digest the peptide bond from the polypeptide chain different enzymes will recognise different peptide bonds after certain side chains to chop them up LSM2106 34  digests the polypeptide chain into small fragments (e.g. tripeptides) in small intestine, there is aminopeptidase (does not have specificity to any side chain groups) → cut all polypeptide chain, break down dipeptides and tripeptides into single amino acids the active proteases are synthesised in the zymogen form (Pro- xxx-ase, xxx-gen) specificity → boils down to 3d configuration of active site, only certain side chains can fit in and be recognised for cutting LSM2106 35  all the zymogens need to have a small piece cut out to activate the enzyme Proteolytic activation of trypsinogen protease needs to cut the peptide bond between lys 15 and ile 16 (by an enzyme called enteropeptidase and trypsin) to form trypsin (active); trypsin will then go back to further cut their own zymogens = autodigestion/ autolysis trypsin can also cut the other zymogens trypsin activation is super impt after trypsin is activated, there will be cascade activation trypsin can also conduct proteolytic activate chymotrypsinogen trypsin will recognise the peptide bond after arg15 once cut, will become pi-chymotrypsin (active but cannot digest dietary protein) pi-chymotrypsin carries out self digestion at Leu13, Tyr146 and Asn148 release two dipeptides becomes alpha-chymotripsin b. Blood clotting cascade of enzymatic activation allows blood clotting to occur rapidly LSM2106 36  zymogen is cut and activated into active protease, which serves as a protease to cut off another zymogen to activate it,… eventually will reach a zymogen that converts into factor Xa (10a) activated factor X forms prothrombinase complex together with Va (5a) (cofactor of protease Xa), will become a fully functional protease which will cut prothrombin (zymogen) into thrombin (protease) thrombin converts fibrinogen into fibrin fibrin will (form polymer strands) cross link with other factors (platelets and RBCs) and aggregate into order filamentous to form the clot 3. Allosteric regulation regulated by molecules called modulators (or effector) that bind non-covalently at a site that is not the active site leads to conformational change composed of multi-subunits presence of modulator (or effector) alters enzyme activity reduced activity/ inhibition → negative modulator activation → positive modulator classification of modulators homotropic modulators → substrate serves as a modulator active site can bind to substrate substrate can also bind to other sites heterotropic modulator → modulator is different from the substrate for example, enzyme has catalytic subunit and regulatory subunit, after modulator has bound to the regulatory subunit, there will be LSM2106 37  conformational change in the catalytic subunit to allow increase/decrease binding of the substrate Allosteric enzyme kinetic profile recall that allosteric enzymes consist of several subunits does not exhibit typical MM hyperbolic curve cooperative substrate binding creates sigmoidal curve instead - binding of substrate to one subunit makes it easier for additional substrate molecules to bind to the other sites each subunit has “crosstalk”, or communication between the binding units The “Km” for allosteric enzyme is K0.5, the substrate concentration at half Vmax in the presence of effector positive effector → curve shifts to the left negative effector → curve shifts to the right Vmax eventually approaches the same value, unaffected by effector Km increases for negative effector and decreases for positive effector At a substrate concentration, order of Vo is positive effector > no effector > negative effector rarely we have affected Vmax and unaffected K0.5 Product inhibition end product of the pathways tends to be the negative effector of the first step of the enzyme reaction building up of the end product ultimately slows the entire pathway Allosteric inhibition of threonine dehydratase by L-isoleucine conversion of L-threonine to L-isoleucine in five steps L-isoleucine functions as heterotropic allosteric feedback inhibtiion LSM2106 38  Feedback inhibition of ATCase regulates Pyrimidine synthesis ATCase catalyses the first step for the synthesis of cytidine triphosphate (CTP) CTP serves as a negative effector and inhibits ATCase (if too much is synthesised) ATP serves as a positive effector and shifts the curve to the left purine: adenosine, guanine Pyramidine: thymine, cytosine balance purine and pyramidine nucleotide amounts when ATP level is higher than CTP, ATCase is activated to synthesise more CTP until the concentrations of ATP and CTP become balanced 4. Covalent modification proteins can be phosphorylated, phosphorylation of hydroxy groups on serine theorine tyrosine, by the enzyme kinase (adds phosphate to protein) this process is reversible by another enzyme phosphatase after the hydroxy groups (from Ser/Thr/Tyr residue) on regulatory subunit is phosphorylated via the action of a protein kinase, there will be a conformational change induced that allows the substrate to bind to the catalytic sites more easily it is also possible for the revere to be true: when the protein is not phosphorylated it is functioning at full capacity, but after being phosphorylated enzyme activity is shut down allosteric regulation of muscle glycogen phosphorylase glycogen is the stored form of glucose when you need energy, you need to hydrolyse the glycogen to release glucose for body to replenish energy Glycogen phosphorylase catalyses the release of glucose units from glycogen LSM2106 39  this enzyme has multiple subunits after adding a phosphate group to glycogen phosphorylase, it becomes glucose 1-phosphate (G1P) G1P of the reaction is converted to G6P which enters glycolysis to generate energy (ATP) G6P and ATP are both products along this pathway when too much ATP, don’t need glycogen phosphorylase activity to be high don’t want to break down extra glucose when the body already has sufficient energy if too much G6P it also means there is accumulation and you want it to slow down G6P and ATP are negative effectors to glycogen phosphorylase (allosteric enzyme) in the presence of ATP and G6P, the curve shifts to the right When the body expends energy (during exercise), ATP will be hydrolysed into ADP and AMP if you have a lot of AMP, it represents that the body is short of energy AMP is a positive effector for glycogen phosphorylase in the presence of AMP, the curve shifts to the left [G6P] and [ATP] high → low energy expenditure → inhibit glycogen phosphorylase → conserve glycogen [AMP] high → cells need energy → glycogen phosphorylase activated → glycogen break down → glycolysis → produce ATP → meet energy need covalent regulation of muscle glycogen phosphorylase Muscle glycogen phosphorylase exists in 2 forms: active a form less active b form LSM2106 40  2 ser14 side chain hydroxy group (less active b)→ can be phosphorylated by phosphorylase kinase → to produce (more active a) glycogen phosphorylase epinephrine, calcium ion and AMP (which are elevated during exercise, for example) stimulate the kinase that phosphorylates the enzyme, increasing the active a form, thus activating the breakdown of glycogen at rest, phosphorylase phosphatase dephosphorylates glycogen phosphorylase and convert it to the less active b form Clinical applications a. Diagnostics a. Measuring plasma enzyme activity during normal cell turnover (death death rate), some of the intracellular enzyme is released into the capillary however, if tissues have damage, increased cell death as a result of disease or trauma causes more intracellular enzyme (and other proteins) to be released into the capillary if certain isoenzymes are predominant in certain tissues, that isoenzyme can be seen as specific biomarker for that tissue Creatine Kinase (CK) as a biomarker for myocardial infarction 3 isoenzymes, each all them composed of two polypeptides (called B and M subunit) CK1 = BB, CK2 = BM, CK3 = MM Skeletal muscle: 98% CK3 and 2% CK2 Cardiac muscle: 70% CK3 and 30% CK2 Other tissues: mainly CK1 can conduct electrophoresis Kinetics of release of cardiac enzymes into serum following a myocardial infarction also measure LDH activity at regular intervals in addition b. aminotransferases and liver damage LSM2106 41  alanine aminotransferase (ALT) aspartate aminotransferase (AST) if present in serum above threshold, it is indicative of liver disease occupational medicine: liver damage generated by toxic solvent monitor liver enzymes when taking some lipid-lowering, anti- diabetic and anti-hypertension drugs c. Couple Assays when the product and substrate of the reaction of interest cannot be easily measured, it is often necessary to couple the reaction to a second reaction for example, if he enzyme of interest is glucokinase, both the product (G6P) and substrate (glucose) don’t have very distinct absorption profile that can be measured by spectrometer So G6P is coupled with G6P dehydrogenase and reacted with NADP to form 6-phosphogluconolactone and NADPH NADPH has an aromatic ring that provides it a unique absorbance at A340 Glucose detection strip (reagents/ enzyme immobilised on a strip) [glucose] high, produce more hydrogen peroxide react with second coupled reaction to show coloured dye b. Analytical tools Enzyme-Linked ImmunoSorbent Assay (ELISA) 1. wells of the plate are coated with antigen of interest 2. wells are then filled with patient’s serum if antibodies against the antigen is present, they will bind to the antigens fixed to the bottom of the well - only antigen-specific antibodies will bind to the wells wells are then washed out to remove unbound anitbodies 3. next, a solution of animal antibody against human anitbodies is added LSM2106 42  this second antibody is covalently conjugated to an enzyme wells are washed again to remove any unbound enzyme- conjugated anitbody 4. finally a solution of a colourgenic enzyme substrate is added the interaction of the substrate with the enzyme on the second anitbody generates visible colour change c. Therapeutic tools drugs: enzyme inhibitors streptokinase streptokinase is a plasminogen activator conversion of plasminogen to plasmin (active form) clear blood clots by stimulating the conversion of plasminogen to plasmin plasmin is a protease that breaks up the molecules of fibrin Abzyme (engineered catalytic antibodies tumors are selectively destroyed while healthy cells are spared from the toxic effect of cancer drugs Has 2 domains: Antibody domain has tumor cell binding site abzyme domain has prodrug activation site that converts inactive prodrug to active anti-cancer drug Abs binds the tumor cells with high affinity Prodrug is introduced into the bloodstream, but only becomes activated in the vicinity of the targetted antibody Lecture 8 - Cellular oxygenation Myoglobin is needed to: prevent Fe2+ group from oxidation to Fe3+ Fe3+ makes heme unable to bind to O2 anymore prevent release of superoxide ion formed (highly reactive and can damage cellular biomolecules) from oxidation of heme (withdrawing LSM2106 43  electrons from Fe2+) decrease affinity of heme for CO due to the perpendicular bonding of CO to the heme plane, it is able to bind to free heme 25000 times better than O2 His E7 makes it such that CO can only bind to heme slanted, due to the steric hindrance of His E7 In fact, since O2 binds to heme in a slanted manner already due to the electron orbitals, the steric hindrance of His E7 does not affect the binding of O2 to heme group CO level in the atmosphere is very low, so even though it binds better, O2 still binds more there exists a resonance structure between Fe2+ and dioxygen and Fe3+ and superoxide ion. His E7 also stabilises the superoxide ion (resonance structure of oxygen-bound heme) so that the superoxide does not leave the Fe in 3+ state(which does not bind O2) The conformational change (”flattening” of heme plane”) brought about by the binding of O2 to heme group does not change the function since it is already bound BUT will affect the function of haemoglobin when heme is not bound to O2, Fe2+ is located above the porphyrin ring plane, towards the direction of HisF8, adopting a “dome” shape when O2 binds, it pulls Fe2+ towards the porphyrin plane and the heme adopts a planar configuration the Fe2+ movement induces a shift in the position of the F helix of myoglobin, which in turn alters myoglobin configuration does not affect myoglobin function but greatly alters the function of haemoglobin hyperbolic oxygen dissociation curve Haemoglobin 1 haemoglobin has 4 heme groups, so it can bind to four O2 in total They bind one by one, binding of one will result in the conformation change of adjacent subunits Oxygen dissociation curve: LSM2106 44  Partial pressure of oxygen is the amount of O2 in the blood → high in the lungs and low in the tissues Wide range of dissociation allows it to transfer oxygen from region of high concentration to region of lower concentration coorperative binding in haemoglobin → sigmoidal oxygen dissociation curve Oxygenation and conformational change O2 pulls the Fe2+ → pulls the F helix (connected to helix E and helix G) deoxyhaemoglobin will cause the subunit to tilt slightly (conformational change) between ap dimer 1 and dimer 2, the interaction would be much stronger, oxygen binding would relax the bonding between the two dimers As oxygenation occurs, it gets increasingly easier. As deoxygenation occurs, it also gets increasingly easier. the dragging of f helix results in conformational change from T state (deoxy) to R state (oxy), which increases O2 affinity in unbound subunits, resulting in more O2 binding = cooperative binding of O2 All these allosteric effectors (protons, CO2, 2,3-BPG) stabilise the T- state, promoting oxygen release Effectors that affect binding of oxygen to hemoglobin (allosteric negative effectors that stabilise the T state, reducing affinity towards O2) 1. Protons As acidity increases, H+ conc increases, affinity of Hb towards O2 decreases, binding of O2 diminished (promotes dissociation of O2) Bohr effect Imagine in the veins (there is respiration producing CO2) you want O2 to be released binding of O2 induces transition induces transition from T to R state which breaks the ionic interaction between His 146 and Asp 94 When blood [H+] increases, equilibrium shifts to the left and stabilises T state LSM2106 45  increase of blood [H+] promotes protonation of His146 and consequently the release of oxygen by favouring transition to T state Metabolism (oxidation of glucose) of tissues produces CO2 that diffuses into red blood cells and generate H2CO3 and thus H+ Red blood cells contain high level of carbonic anhydrase that accelerates H2CO3 production (CO2 + H2O → H2CO3) H2CO3 then dissociates into HCO3- (bicarbonate) and H+ Increase of blood [H+] around body tissues promotes protonation of His 146 → favour transition to T state → release of O2 2. Carbon dioxide R-NH2 (N-terminus of a2 chain) + CO2 → R-NH-COO-(carbamate) + H+ CO2 directly reacts with N-terminus of globin chain (of haemoglobin) to form carbamate ion carbamate ion interacts with a positively charged Arg side group, which forms an ionic interaction that stabilises T-state, promoting release of O2 this ionic interaction is only established in the T-state when haemoglobin adopts R-state, the carbamate ion is not stabilised and is released as CO2 Carbonic anhydrase activity (in prev part) Putting it all together: High pO2 in lungs enhances R-state in haemoglobin (equilibrium of Hb + O2 ⇌ HbO2 shifts to the right) The R-state destabilizes the ionic interactions and carbamate ions. Thus, H+ and CO2 are released from oxyhemoglobin When haemoglobin (in red blood cells) travels to extra- pulmonary tissues, such as skeletal muscles, O2 is released due to low pO2 (equilibrium of Hb + O2 ⇌ HbO2 shifts to the left) Release of O2 changes haemoglobin from the R- to T-state, which enhances binding of H+ (to His 146) and CO2 (as LSM2106 46  carbamate ion) to deoxyhemoglobin, and the resultant ionic interactions further stabilizes the T-state. 3. Metabolite 2,3-bisphophoglycerate (2,3-BPG) 2,3-BPG is the most abundant organic phosphate in the red blood cell, synthesised from an intermediate of the glycolytic pathway it can nicely fit into the positively charged cavity formed by the inward-forming negative charges in the deoxy form Hb, stabilising T- state 2,3-BPG only binds to the T form (deoxy) In the absence of BPG, oxygen binding follows a hyperbola → no more corporative binding(?) In high altitudes, the binding gets even weaker, and curve shift to the right increase amount of 2,3-BPG synthesised in RBC greater unloading of O2 in the capillaries of tissues Fetal hemoglobin (HbF) gamma subunit is very similar to beta subunit but has ser instead of his at position 143, thus lacking two of the positive charges in the central BPG binding cavity 2,3 -BPG bind weaker to HBF because of the replacement of histidine to serine (which lacks the positive charges) HbF curve shifts to the left (under the same pO2, the fetal red blood cells will have a higher saturation fragment) → higher affinity for O2 fetus haemoglobin needs to have a higher affinity than the mother in order for the oxygen to flow from the mother to the fetus to ensure the fetus has sufficient supply of oxygen Sickle-cell Anemia (HbS) caused by a point mutation in B-haemoglobin Glu to Val mutant Val side chain in the HbS fits into a hydrophobic pocket on the surface of a B subunit in another HbS tetramer LSM2106 47  sickled cells can block blood flow and die faster (blood has less blood cells than normal) In the sickle cell there is a conformation change as it converts from oxy to deoxy, there is now a hydrophobic pocket (only in T form), and the protrusion in the cell (as a result of mutation to Val) can now fit into the pocket and cause agglomeration of cells hydrophobic interactions between the mutant Val of the B subunit and hydrophobic pocket of a neighbouring hemoglobin initaties the aggregation of deoxyhaemoglobin hydrophobic pocket will be gone in oxygenated blood (R form) → HbS fibers dissolve essentially instantaneously upon oxygenation, thus almost absent in arterial blood Lecture 9 - Carbohydrate metabolism Overview of glycolysis glycolysis → breaking down of glucose (6C) into pyruvate (3C) ATP is needed, but there is a net gain of ATP NADH is also produced, it serves as an electron carrier glycolysis that occurs in the cytoplasm does not require oxygen citric acid cycle that occurs in the mitochondria requires oxygen When oxygen is present, pyruvate will enter the mitochondria. first it will react with enzyme A to form acetyl CoA, which enters the Kreb’s cycle/ TCA cycle A lot of NADH is generated in this step Electron transport chain transmembrane proteins NADH will transfer the electrons to these protein complexes, which will eventually reach oxygen (final electron acceptor) to form H2O, NADH is oxidised and energy is released, energy released is used to pump protons from the matrix into intermembrane space to generate pH gradient, so that when the protons flow back into the matrix through ATP synthase, energy will be used to generate ATP molecules LSM2106 48  oxidative phosphorylation (ADP is being phosphorylated, while NADH is being oxidised) occurs at the inner membrane of the mitochondria In the case of prokaryotes, this process occurs the plasma membrane Molecules to take note of: Glucose-6-phosphate Aldehyde-3-phosphate These are products that are formed from other processes, that are able to join into the glycolysis reaction Redox reactions Oxidation: loss of electrons/ oxidation state increases Reduction gain in electrons/ oxidation state decreases NAD+ + H+ + 2e− ↔ NADH NAD+ is the oxidised form which undergoes reduction and acts as the oxidising agent NADH is the reduce form which undergoes oxidation and acts as the reducing agent this is the state that carries the high energy electron, so they contain high amount of energy comparatively Energy is released for oxidation but energy needs to be provided for reduction for it to reach the higher energy state Glycolysis process that happens regardless of whether oxygen is present or not - oxygen independent it is the first step in carbohydrate metabolism energy first needs to be provided: glucose activation step you need to provide some energy first before you can activate the glucose (by phosphorylating it using 2 ATP molecules) to break it down into a 3C molecule (each carrying 1 phosphate group) LSM2106 49  4ATP is produced (so net gain of 2 ATP) and 2 NAD+ is reduced to 2 NADH, forming 2 pyruvate molecules glycolysis is 100x faster than oxidative phosphorylation in ATP production what happens after glycolysis when there is insufficient oxygen (anoxic regeneration of NAD+ in glcolysis, or anaerobic glycolysis) no/ insufficient oxygen available to go into aerobic respiration enters a process known as lactate fermentation if you break down too much glucose you will run out of NAD+ (as it is needed in glycolysis) so you need to reconstitute back the NAD+ before you can continue breaking down more glucose pyruvate will be reduced by NADH (reducing agent) with the help of lactate dehydrogenase (NADH as the cofactor) to form lactate/ lactic acid, while NADH is oxidised into NAD+ (which is available to break down more glucose to generate more ATP) A lot of lactic acid will be generated, if sustained for long, tissue/ muscles also become more and more acidic, the accumulation of which inhibits lactate dehydrogenase (environment is too acidic for function), this process is not sustainable lactate is the reduced form of pyruvate, so it also contains a fairly high amount of energy the lactate will be stored in muscle when the body regains sufficient oxygen, the lactate can be converted back into pyruvate, then back into glucose (gluconeogenesis) convert high energy lactate back into glucose which can be used for aerobic respiration, it will not be wasted when this fermentation occurs in bacteria, it will produce a lot of lactic acid (making of yoghurt, bacteria ferment a lot of lactic acid in anaerobic conditions to curd the protein in the milk) in yeast (eukaryotic), pyruvate is first converted to acetaldehyde with the help of pyruvate decarboxylase, releasing CO2 in the process; followed by reduction into ethanol with the help of alcohol dehydrogenase and NADH (converted back to alcohol) ⇒ alcohol fermentation (making of beer and wine) LSM2106 50  lactate fermentation is dangerous if it happens in the brain, will causes permanent damage to brain tissue (is the reason why stroke is so dangerous, because some regions of the brain will not have supply of oxygen, and brain cells enter fermentation and produce a lot of lactic acid which damages brain tissue) Formation of Acetyl-Coenzyme A (Acetyl-CoA) in aerobic condition, pyruvated is transported into the mitochondrial matrix pyruvate (3C) will have CO2 removed, and will react with coenzyme A, to form Acetyl CoA (2C) During this process, energy is used to reduce NAD+ to form 1 NADH molecule pyruvate + coenzyme A + NAD+ → Acetyl CoA + CO2 + NADH + H+ oxygen is not needed for this process this process does not generate any ATP, but will generate one NADH and CO2 Acetyl CoA will then enter the tricarboxylic acid (TCA) cycle Tricarboxylic acid (TCA) cycle also happens in the mitochondrial matrix named after citric acid (has 3 carboxylic acid groups) as it is the starting and ending products of this cycle oxidative decarboxylation (6C → 5C → 4C), each step has 1 CO2 removed, and generate NADH molecule at each step Succinyl CoA (4C) undergoes substrate level phosphorylation to generate 1 ATP, then goes thru another 2 dehydrogenation steps (first one generates 1 FADH2 and the next one generates NADH) finally, the remaining 4C molecule (oxaloacetate) will be combined with acetyl CoA (2C) to return to the 6C state (citrate) for each Acetyl-CoA that enters the cycle, you will generate 2CO2, 3NADH, 1 FADH2, 1ATP (each glucose will produce 2 acetyl CoA so x2 cycle) FADH2 is also another electron carrier in the reduced form LSM2106 51  Transfer of NADH into mitochondria matrix remember that you still have 2 NADH molecules that were generated in the cytoplasm during glycolysis, which will be transported into the mitochondria (as they also carry high amt of energy) to transport NADH in the cytoplasm into the mitochondria, we have a shuttle pathways malate-aspartate shuttle is the principal mechanism for the movement of NADH (from glycolysis) from cytoplasm into mitochondria matrix in adults aspartate can be converted into oxaloacetate, and NADH is used to transfer electrons (becomes NAD+) to oxaloacetate to form malate, which is able to enter the mitochondria, then it is oxidised back into oxaloacetate by NAD+ (which is reduced to NADH) and then back into asparate which leaves the mitochondria the cycle continues all the NADH from outside the mitochondria will be transferred to the inside of the mitochondria glycerol-phosphate shuttle dihydroxyacetone phosphate reacts with NADH to be reduced into glycerol 3-phosphate when glycerol 3-p is returning into hydroxyacetone phosphate, the electron will be used to reduce FAD to FADH2 (reduced form) FADH2 contains a smaller amount of energy compared to NADH, lesser energy produced this shuttle is mainly found in brown adipose tissue in infants, as the conversion is less efficient, thus generating heat to keep warmth this glycerol phosphate shuttle is a secondary mechanism for transport of cytosolic NADH to mitochondrial matrix for adults Electron Transport Chain (ETC) and Oxidative Phosphorylation NADH and FADH2 in mitochondrial matrix are high energy electron carriers LSM2106 52  electron transport chain through a series of integral multiprotein complexes at the inner membrane of mitochondria (ETC) to an electron acceptor (oxygen is the final electron acceptor, which will be reduced to form water) this transfer of electron through the ETC releases energy the energy is used to pump protons out of the matrix into the intermembrane compartment (complex 1,3,4 each pump 4 protons) to generate a pH gradient when the protons diffuse out of the intermembrane compartment back into the matrix, ATP is synthesised in the process of oxidative phosphorylation through the action of ATP synthase (rotates counterclockwise, 1 ATP comes in 1 goes out, repeats) each 4 proton will be able to generate 1 ATP each NADH (12H+ is pumped out into intermembrane space) will be able to generate 3 ATP molecules but since FADH2 is only enters the ETC in complex 2 (8H+ is pumped out into intermembrane space), it is only able to generate 2 ATP molecules Sometimes, the oxygen turns into superoxides (instead of water) when receiving the electrons at the end of the ETC these are reactive oxygen species (ROS) will also be generated by oxidative phosphorylation ROS will cause DNA damage, cell death and aging superoxide dismutase is needed to remove ROS Overall 1 glucose → 2 pyruvate + 2 ATP 2x (pyruvate → acetyl CoA + NADH) TCA cycle (for 2 pyruvate/ 1 glucose): 6 NADH + 2 FADH2 + 2 ATP From NADH malate-aspartate shuttle: additional 2 NADH from cytoplasm Total: 10 NADH, 2 FADH2, 4ATP Total: 30ATP + 4 ATP + 4ATP = 38 ATP (36 if using glycerol-phosphate shuttle, because 2 NADH is converted to 2FADH2, which each produce 1 LSM2106 53  less ATP) This is a lot compared to glycolysis alone, which can only produce 2 ATP from 1 glucose Breaking down of glycogen (glycogenosis) glucose amount in the bloodstream is highly regulated storage system for these carbohydrates when there is too much glucose, it can be stored as glycogen when there is too little glucose, it can be released glycogen is a polysaccharide, a polymer composed of many glucose monomers glycogen (liver sugar) is composed of glucose molecules linked by a- 1,4-glycosidic bonds, and a-1,6-glycosidic linkages at branch points (a- 1,6-glycosidic linkages cause branching from linear structure) The breaking of glycogen bonds to release glucose is known as glycogenolysis glycogen phosphorylase catalyses cleavage of a-1,4-glycosidic bond by phosphorylysis, releasing glucose-1-phosphate glycogen phosphorylase adds a phosphate group to the 1C of the glucose subunit to cleave a-1,4,-glycosidic bond to produce glucose-1- phosphate molecule glycogen (n residues) + Pi → glycogen (n-1 residues) + glucose-1- phosphate However, this only occurs up until 4 residues of an a-1,6-linkage. This reaction can only start from a linear strand - it cannot go too close to the branch. This is called “limit branch” Debranching enzyme is needed to remove 3 glucose units from the branch to put it back to the linear chain has two independent active sites, one for a-1,6-glycosidase activity to break down the a-1,6-glycosidic bond and the other for transferase activity transferase transfers 3 glucose residues from a 4-residue limit branch to the end of another branch, diminishing the limit branch to LSM2106 54  a single glucose residue The single glucose residue is removed using a-1,6-glucosidase to catalyse hydrolysis of the a-1,6-linkage, yielding free glucose glucose-1-phosphate cannot be used directly for metabolism, needs to be converted back to glucose-6-phosphate enzyme is needed to move the phosphate group from position 1 to position 6 phosphoglucomutase catalyses the reversible reaction to generate glucose-6-phosphate, which can enter glycolysis OR can be dephosphorylated to glucose by glucose-6-phosphatase (mainly in liver) Gluconeogenesis - glucose from non-carbohydrate sources gluconeogenesis is a metabolic pathway that generates glucose from certain non-carbohydrate carbon substrates for example, glucogenic aminno acids (from protein), glycerol (from lipid), pyruvate and lactate (from anaerobic glycolysis) Lactate molecules contain a lot of energy, can be converted back to glucose through the cori cycle. Cori cycle involves utilisation of lactate (in muscle and RBC) as a carbon source for hepatic gluconeogenesis. Lactate dehydrogenase converts lactate back into pyruvate in hepatocyte through redox reaction pyruvate can undergo gluconeogenesis to go back into glucose. However, the conversion of 2 pyruvate to 1 glucose will consume 6 ATP. This is very energy costly and is an energy expensive process, so the process cannot last forever Energy needs to be provided before you can convert these non- carbohydrates substances back into glucose Gluconeogenesis is a mechanism used to maintain blood glucose level, avoiding hypoglycemia, especially in the brain tissues that can only use glucose or ketone bodies as energy sources, and cannot use lipid. The cells will still need to convert these substances into glucose so that the glucose can be used by the brain, even if this is an energy expensive process Formation of glycogen (glycogenesis) LSM2106 55  too much glucose in the bloodstream - diabetes - can damage blood vessels and make blood too viscous and change osmolarity of blood convert glucose into glycogen G6P → G1P Uridine triphophate (UTP) react with G1P with the help of UDP-glucose phosphorylase to form Uridine diphosphate glucose (UDP glucose) +2Pi Glycogenin has tyrosin residues on the surface of the protein, this tyrosine can react with UDP, UDP will be removed, C1 of glucose join to oxygen of OH group of tyrosine, forming a glycosidic bond. Glucose will be linked to glycogenin, more UDP glucose will continue to be added, remove the UDP, and join another one, until you have a short chain of glucose molecules linked to glycogenin glycosidic bond is formed between the anomeric C1 of the glucose moiety in UDP-glucose and hydroxyl oxygen of a tyrosine residue in glycogenin with the release of UDP glycogenin then catalyse glucosylation at C4 of the attached glucose using UDP-glucose as a glucose donor. This process is repeated until a short linear glucose polymer is built up Glycogen synthase catalyses elongation of glycogen chains glycogen (n residues) + UDP-glucose → glycogen (n+1 residues) + UDP branching enzyme transfer a segment from the end of a glycogen chain to the C6 hydroxyl of a glucose residue of glycogen to yield a branch with a-1,6-linkage glycogen is insoluble and compact, allow storage of large amount of energy without upsetting the osmotic pressure of the cell or affecting blood glucose level Disease conditions with carbohydrate metabolism 1. Type 1 galactosemia problem digesting lactose (lactose intolerance) - lactose is a disaccharide comprised of galactose and glucose in order for galactose to be digested, you need to convert them into something else before you can utilise galactose LSM2106 56  first need to convert galactose to galactose-1-phosphate with galactokinase(GALK), then go through GALT (galactose-1-phosphate uridyltransferase) enzyme to transfer a UDP onto galactose 1 phosphate, to create a UDP-galactose. UDP-galactose will then be converted by epimerase (GALE) into UDP glucose. UDP-glucose will be able to form glucose 1 phosphate, which is finally converted to glucose-6-phosphate, that can undergo glycolysis but in some people the activity and concentration of the GALE protein is very low, leading to accumulation of galactose (that cannot be converted into glucose) Type 1 galactosemia arises from deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT) when these people consume milk, because they cannot digest galactose, they will experience diarrhoea and vomiting, and will need to drink lactose-free milk caused by a single mutation (most commonly a substitution of glutamine for arginine at amino acid 188), close to the active site of GALT 2. Hereditary fructose intolerance fructose is mostly found in fruits to digest fructose, it needs to first be converted into fructose-1- phosphate using ATP; fructose-1-phosphate is broken down by aldolase B (in the liver) into glyceraldehyde-3-phosphate (recall that this is a component in the glycolysis pathway), which enters glycolysis hereditary fructose intolerance (HFI) is resulted from a lack of aldolase B in the liver, small intestine and kidney patients don’t have anough aldolase B fructokinase (adds phosphate groups onto fructose, 1st step of digesting fructose), lacks feedback inhibition and continues rapidly phosphorylates fructose to form fructose-1-phosphate by transferring ATP to fructose. This uses up a lot of ATP, leading to consequent depletion of ATP. This also traps a lot of phosphate (Pi) into fructose-1-phosphate LSM2106 57  Phosphate groups are very important. Loss of Pi impairs glycogen breakdown. Because there is not enough phosphate, even if there is a lot of glycogen, it will not be able to be broken down This will cause hypoglycemia (low amount of glucose in the blood because you cannot break down glycogen), and vomiting upon ingestion of fructose or sucrose Objective 1: Differences between anaerobic and aerobic glycolysis Objective 2: How ATP is generated through a series of redox reactions Objective 3: What happens when there is too much or too little glucose Objective 4: Diseases caused by dysfunction of carbohydrate metabolism Lecture 10 - Carbohydrate as cellular component Carbohydrates chemistry and nomenclature composed of C,H,O mainly classified into two classes: aldose and ketose, depending on the kinds of groups found on the carbonyl carbon aldose: aldehyde group (RCHO) ketose: ketone group (RC=OR) monosaccharides are named based on whether it contains an aldehyde or ketone group at the carbonyl carbon and also the total number of carbon atoms terminal CH2OH group or alcohol group Central -OH or hydroxyl groups Stereoisomers of monosaccharide chiral carbon atom (chiral centre) has 4 different connections (connected to 4 different groups) chiral centres allow for formation of stereoisomers number of possible stereoisomers = 2n (n = number of chiral centres) Enantiomer (L or D form, mirror images) whether it is the D form or L form is decided by the position of the - OH group at the chiral carbon furthest away from the carbonyl group LSM2106 58  right = D; left = L D form is the major/ predominant form Stereoisomers that not mirror images of each other are called diastereoisomers Epimers: diastereoisomers that differ in 1 position, e.g. mannose (C2) and galactose (C4) are epimers of glucose D-aldose stereoisomers D-ketose stereoisomers LSM2106 59  Cyclisation of monosaccharides monosaccharides usually exist as cyclic form in solutions Aldose C5 -OH group nucleophilic attack carbonyl group of aldehyde (C1), breaking the double bond and shifts H of hydroxyl group on C5 to carbonyl group oxygen, forming a cyclic form C1 (position originally occupied by carbonyl) is now known as the anomeric carbon This anomeric carbon allows for 2 stereoisomers (anomers) The position of OH on C1 decides whether is is: alpha (OH below ring, different side as the OH group on C6), or LSM2106 60  beta (OH above ring, same side as the OH group on C6) anomer six-sided ring is called a pyranose. 99% monosaccharide exist in cyclic form Ketose Carbonyl/ Ketone group is at C2 (rather than in C1 for aldose) C5 -OH group attack carbonyl group of ketone (C2), breaking the double bond and shifts H of hydroxyl group on C5 to carbonyl group oxygen C2 (from carbonyl of ketone group) is now the anomeric carbon The position of OH on C2 decides whether it is: alpha (OH below ring), or beta (OH above ring) anomer Five-sided ring is called a furanose (but it still has 6 carbons) Types of isomers Constitutional isomers: differ in the order of attachment of atoms/ functional groups Stereoisomers: atoms are connected in the same order but differ in spatial arrangement Enantiomers: nonsuperimposable mirror images Diastereoisomers: nonsuperimposable non-mirro images Epimers: differ at one of several asymmetric carbon atoms LSM2106 61  Anomers: isomers that differ at a new asymmetric carbon atom formed on ring closure Derivatives of monosaccharides change the chemical nature of the sugar esterification: adding phosphate to form sugar ester OH removal: deoxy-sugar reduction: sugar alcohol oxidation: sugar acid amination: amino sugar the most impt 2 are amino sugar and sugar acid, as they can combine to form GAG (glycosaminoglycan), a component of proteoglycan Disaccharide 2 monosaccharides -OH group at the anomeric carbon (reducing end) react in a dehydration reaction with the -OH group from another sugar to form a glycosidic bond the glycosidic bond can be broken by hydrolysis the glycosidic bond is named on its anomeric forms (a or b) and the positions of the carbon atoms forming the bond LSM2106 62  Polysaccharide 1. Starch and glycogen - storage of glucose homopolysaccharide (every unit is the same (glucose)) amylose (aka starch) and amylopectin (aka glycogen) LSM2106 63  human can digest a-1,4-glycosidic bond but not b-1,4-glycosidic bond hence why humans can digest amylose but not cellulose debranching enzyme is needed for glycogenolysis used for storage of glucose starch in plants glycogen in animals depending on the types of bond, it will give rise to different structures a-1,4 linkage is bent, so it will not exist as a linear chain if all the linkages between units are bending in the same direction starch exist in a a-helical structure because of all the bents at a- 1,4-glycosidic bonds allows starch to be stained by iodine, because iodine can insert into the middle of the amylose helix, leading to blue colouration breakdown of starch/ digestion of amylose chain by a-amylase (in saliva) using hydrolysis 2. Cellulose - structural material glucose connected by b-1,4 glycosidic bonds (terminal CH2OH groups of adjacent units are opposite sides of the chain) → forms a linear chain (instead of helical structure) intra-chain hydrogen bonds formed → linear chain inter-chain hydrogen bonds formed by -OH groups of the ring with adjacent chains to form a sheet LSM2106 64  sheets can be stacked upon each other Inter-sheet hydrogen bonds can be formed between the alternating - CH2OH group above and below the plane starch, on the other hand, has all the CH2OH group on one side of the plane Cellulose used as structural materials in plant Humans don’t have enzyme to break b-1,4-glycosidic bond, cannot gain glucose from cellulose Bacteria in the intestine of cow secrete cellulase to digest b-1,4 glycosidic bonds in cellulose, cow can get carbohydrates from grass 3. Glycosaminoglycan (GAG) - heteropolysaccharide of modified sugar multiple monosaccharides are used amino sugar and sugar acid combine to form a disaccharide that is repeated for many times to form a long polysaccharide chain the disaccharide unit contain either of two amino sugars, GalNac or GlcNac, and an uronic acid such as GlcA and IdoA amino sugar has a NH2 group that is acetylated -OH group is above the ring = galactose; -OH group is below the ring = glucose LSM2106 65  glucuronic acid = COO- above; a-L-iduronate = COO- below heteropolysaccharide with repeating disaccharide form a long and unbranched chain composed of amino sugars and sugar acids that carry sufate and carboxylate side chains surfaces are very negative, able to attract metal ions that carry water Highly negatively charged (attract positive ions and water) and can be linked to protein to form proteoglycan if a molecule contain a lot of GAG, the solution will be highly viscous and trap a lot of water, making them have low compressibility in solution, can be used as lubricating fluids in joints GAG located mostly on cell surface (membrane bound) or in the extracellular matrix (ECM) or cartilage Examples: LSM2106 66  hyaluronic acid does not contain sulphate and not covalently attached to protein. It is a component of non-covalently formed complexes with proteoglycan in ECM. Hyaluronans are the largest polysaccharide (MW of 100,000 - 10,000,000) produced by vertebrate cells Dermatin sulfate fuunction in coagulation, wound repair, fibrasis and infection Chondroitin-4-sulfate is associated with protein to form proteoglycans, e.g. aggrecan, which is a major component of cartilage Heparin is an anti-coagulant (clinically) but function to defend against invading bacteria and foreign substances in vivo. Heparan associated with proteins to form heparan sulfate proteoglycan (HSPG), which binds fibroblast growth factors (FGFs); vascular endothelial growth factors (VEGF); and hepatocyte growth factors LSM2106 67  Keratan sulfate associated with protein to form proteoglycans, e.g. aggrecan disaccharides repeating many times, with many forms of acids and sugars linked together Carbohydrate linked to peptide, protein and lipid Carbohydrate linked to peptide glycopeptide (peptide constitutes majority of the molecule, carbohydrates is the minor component) not really found in the human body, not a focus of this module peptidoglycan (sugar constitutes majority of the molecule, peptide is the minor component) Peptidoglycan in bacterial cell walls mainly composed of carbohydrates, carbohydrate chains are linked together by peptide peptidoglycan (polysaccharide linked to peptides) play an imporant structural role in bacterial cell wall gram positive bacteria have thick peptidoglycan outer surface of plasma membrane gram negative bacteria have thin peptidoglycan layer in between two layers of lipid bilayer, on top of the lipid bilayer is lipo- polysaccharide (contains carbohydrates (major component) and lipids, but is DIFFERENT from glycolipid) the peptidoglycan layer is substantially thicker in gram-positive bacteria (20-80 nm) than in rgam-negative bacteria 97-8 nm) peptidoglycan constitutes 90% dry weight of Gram +ve bacteria but only 10% of gram -ve strains polysaccharide chain is a heteropolysaccharide, using two types of amino sugars (GlcNAc & MurNAc) MurNAc has carboxylic acid group attaches outward of the position 3 carbon (amino sugar acid?) alternating residues of NAG and NAM will be linked together by B-1,4 glycosidic bonds to form a long heteropolysaccharide LSM2106 68  chain each NAM is attached to a short tetrapeptide of L-alanin, D- glutamic acid, L-lysine and D-alanine the tetrapeptide is cross-linked by a penta-glycine inter-bridge in gram +ve bacteria and by direct linkage in gram -ve bacteria many chains link together to form a sheet, which can be stacked upon each other to form many layers of sheet, that can form strong structural material penicillin is an anitbiotic that kills bacteria formation of peptidoglycan requires an enzyme, DD- transpeptidase, that forms the linkage between the pentaglycine linker and tetrapeptide LSM2106 69  DD-transpeptidase crosslinks the oligopeptides in peptidoglycan penicillin interferes with the production of peptidoglycan by binding to bacterial DD-transpeptidase this prevents the cross linking of the carbohydrate chains, so the bacteria will not be able to form (good) peptidoglycan, and die when they are unable to protect themselves using their cell wall antibiotic resistance results from mutations of bacterial DD- transpeptidase that leads to a reduced interaction with an antibiotic Carbohydrate linked to protein glycoprotein (protein constitutes majority of the molecules, carbohydrates is the minor component) protein (major component) and carbohy