Biochemistry Notes: 2024-2025 PDF

Biochemistry Lesson & book notes By Carolina 2024-2025  Lesson 1: Amino Acids A.A are the building blocks of proteins Composed of a Amino group, Carboxylic group, an -Carbon and a side chain (R-group) The R-group is responsible for the variation of the α-amino acids A...

Biochemistry Lesson & book notes By Carolina 2024-2025  Lesson 1: Amino Acids A.A are the building blocks of proteins Composed of a Amino group, Carboxylic group, an -Carbon and a side chain (R-group) The R-group is responsible for the variation of the α-amino acids Amino acids are zwitterionic At low pH all groups are completely protonated At neutral / specific pH they form dipolar zwitterions  act as buffers At high pH all groups donate their protons Low pH  Neutral pH  High pH  Amino acids are zwitterionic Amino acids carry a net charge of zero at a specific pH (the pI) Zwitterions predominate at pH values between the pKa values of the amino and carboxyl groups For amino acids without ionizable side chains, the Isoelectric Point (equivalence point, pI) is At this point, the net charge is zero AA is least soluble in water AA does not migrate in electric field Around their iso-electric point, amino acids act as buffers as they can donate/ take up protons  Amino Acids: Classification Nonpolar, aliphatic Glycine, Gly, G Alanine, Ala, A Proline, Pro, P Valine, Val, Leucine, Leu, L Isoleucine, Ile, I Methionine, Met, M Glycine not chiral Proline has an imino group (the amino group is held in a rigid conformation). Non-polar a.a are hydrophobic  Amino Acids: Classification Aromatic Phenylalanine, Phe, F Polar, uncharged Tyrosine, Tyr, Y Tryptophan, Trp, W Serine,Ser, S Threonine, Thr, T Cysteine, Cys, C Asparagine, Asn, N Glutamine, Gln, Q These amino acids side chains can form hydrogen bonds. Cysteine can form disulfide bonds (cystine  Amino Acids: Classification Positively charged Negatively charged Lysine, Lys, K Aspartate, Asp, D Arginine, Arg, R Glutamate, Glu, E Histidine, His, H Negatively charged R groups are negatively positively charged R groups are positively charged if the pH is … the pKr value charged if the pH is below the pKr value;  Chirality of amino acids (except Gly) Many molecules and others are achiral identical to its mirror images Not chiral Proteins only contain L amino acids  Practice 1. The rest groups of which amino acids can have a positive charge ? 2. The rest groups of which amino acids can have a negative charge? 3. Which amino acid rest groups are polar without a charge ? 4. Which amino acid rest groups have an amide group; what is the difference with an amino group ? 5. Which rest groups bear a charge at pH = 7 ? 6. What is a motif & domain and what are the differences between them? 7. What is a multimer, an oligomer & a protomer (differences)?  Lesson 2: Peptides & proteins 1. Primary structure: Formation of peptides Peptides are small condensation products of amino acids Water comes free: condensation reaction Peptides are written beginning with the N-ternimal residue which by convention is placed at the left. The side-groups of two consecutive residues are facing opposite directions The C=O – N-H (peptide bond) has a double bond “character”, that is, rigid and not flexible Primary structure: the local bending of the chain The six atoms around the peptide bond lie in a single plane The Ca‘s are the HINGES for the planes. φ (phi): angle around the -carbon—amide nitrogen bond ψ (psi): angle around the  -carbon— carboxyl carbon bond In a fully extended polypeptide, both ψ and φ are 180° Some φ and ψ combinations are very unfavourable because of steric hindrance of backbone atoms with other atoms in the backbone or side chains Some φ and ψ combinations are more favourable because of chance to form favourable H-bonding interactions along the backbone Primary structure: Possible secondary structure A Ramachandran plot shows the distribution of φ and ψ dihedral angles that are found in a protein The areas shaded dark blue represent conformations that involve no steric overlap if the van der Waals radii of each atom are modeled as a hard sphere and that are, thus fully allowed. Medium blue indicates conformations permitted if atoms are allowed to approach each other by an additional 0.1 nm, a slight clash. The lightest blue indicates conformations that are permissible if a very modest flexibility (a few degrees) is allowed in the ω dihedral angle that describes the peptide bond itself. The white regions are conformations that are not allowed.  Secondary structures Secondary structure refers to a local spatial arrangement of the polypeptide backbone Two regular arrangements are common: The  helix: stabilized by hydrogen bonds between nearby residues The  sheet: stabilized by hydrogen bonds between adjacent segments that may not be nearby Irregular arrangement of the polypeptide chain is called the random coil (doesn’t properly describe the structure of these segments, the polypeptide backbone is not random but highly specific) -helix Rodlike structure with the tightly coiled backbone forming the inner part of the rod and the side chains extend outward in a helical way. All a helices found in proteins are right-handed The structure is stabilized by hydrogen bonds between the –NH- and the –CO- groups of the main chain Proline is also an a-helix breaker because it lacks an NH- group. Serine, aspartate and asparagine because their side chains contain hydrogen bond donors or acceptors in close proximity to the main chain  Secondary structure: β-sheet Pleated sheet-like structure: Side chains standout from the sheet alternating in up & down direction A beta-sheet is formed by lining two or more beta-strands lying next to one another through hydrogen bonds. The strands can run in opposite directions (antiparallel) or in the same (parallel).  Secondary structure: loops Loops connect a-helices & b-sheets, usually realize a change in direction Loops are not well defined, hydrophilic and on the surface of proteins Loops consisting of only 4-5 amino acids forming internal hydrogen bonds are called turns Secondary structure: β turns -turns occur frequently whenever strands in -sheets change the direction The 180° turn is accomplished over four amino acids The turn is stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence Proline in position 2 or glycine in position 3 are common in -turns Tertiary structure The overall three-dimensional structure of a polypeptide is called tertiary structure. The tertiary structure is primarily due to interactions between the R groups of the amino acids that make up the protein. The tertiary structures of proteins are formed by the various non- covalent interactions & the disulfide bonds  Non-covalent interactions examples Hydrophobic effect: Complex phenomenon associated with the ordering of water molecules around nonpolar substances Hydrogen bonds Van der Waals interactions (London dispersion): Medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein Dipole-dipole interactions: involving the permanent electric dipoles derived from the fluctuations of the electron cloud surrounding any atom. Electrostatic interactions (ion interactions): Long-range strong interactions between permanently charged groups, or between the ion and a permanent dipole; Salt-bridges, esp. buried in the hydrophobic environment strongly stabilize the protein. Disulfide bond: covalent interaction of the R-groups The number of disulfide bonds defines the protein (eg. fiber) properties. Hair and wool having fewer cross-links are flexible. Horns, claws and hooves having more cross-links are much harder.  Quaternary structure Proteins with more than one polypeptide chain exhibit a fourth level of structural organization Quaternary structure: Refers to the spatial arrangement of subunits (chains) and the nature of their interactions How do proteins reach their three- dimensional structure in a cell? Proteins fold into conformation of lowest energy Determined by amino acid sequence (as demonstrated by spontaneous refolding after denaturation) Conformation of lowest energy is sometimes only marginally stable  allows biological flexibility  Chaperons help proper folding of newly synthesized proteins Hsp: Heat shock proteins; The chaperones do not actively promote the folding of the substrate protein, but instead prevent aggregation of unfolded peptides Denaturation: Loss of structural integrity with accompanying loss of activity Denaturation agent Molecular principle Heat or cold Hydrogen bonds/van der Waals interactions destroyed due to molecular movement (heat); Water molecules enter protein (cold denaturation) pH extremes Mainly disruption of electrostatic (ion) interactions Organic solvent Interacts with side chains and therefore disrupts interaction of side chains among each other Chaotropic agents Induce chaos in water (less hydrogen bonding) – water (e.g. urea and interacts with protein and can disturb hydrophobic guanidinium interactions in protein hydrochloride)  Lesson 3: PTMs & protein targeting How can proteins reach their final destination in or outside of a eukaryotic cell?  Signal sequences are ‘postal codes’ of proteins Signal sequences direct secreted proteins to the Endoplasmatic Reticulum (ER) in the 1 st place Proteins with these signal sequences are synthesized in the ribosomes attached to the ER: 1) Initiation of protein synthesis on free ribosomes 2) The signal sequence appears early (at the N-terminus) 3) Signal recognition particle (SRP) is bound to the ribosome. 4) SRP binds GTP & halts elongation. The GTP bound SRP directs the ribosome & the incomplete polypeptide to GTP bound SRP receptors in the cytosolic phase of the ER. 5) SRP dissociates from the ribosome accompanied with hydrolysis of the GTP in both SRP & SRP receptor. 6) Elongation of the peptide now resumes until the complete protein has been synthesized. 7) The signal sequence is removed.  Secreted proteins are packed into vesicles & transported through ER: Golgi: Removal of signal sequences O-glycosylation Folding Further modification S-S bonds Shortage & delivery Glycosylation to final destinations In the ER newly synthesized proteins are further modified in several ways. Following the removal of signal sequences, polypeptides are folded, disulfide bonds formed & many proteins glycosylated to form glycoproteins (Asn residues). Suitably modified proteins can now be removed to a variety of intracellular destinations. Proteins travel from the ER to the Golgi complex in transport vesicles. In the Golgi complex oligosaccharides are O-linked to some proteins, and N- linked are further modified. The Golgi complex also sorts proteins & sends them to their final destinations. The processes that segregate proteins must distinguish these proteins on the basis of their structural features (other than the signal sequences which were removed in the ER lumen).  Modified amino acids in proteins Permanent post-translational modification of amino acids Glycosylation provides even higher structure variability and enhances solubility of proteins Addition of sugar residues starts in ER; continues in Golgi apparatus Targeting of nuclear proteins Nuclear proteins are recognised & transported by transport proteins Signal sequences for nuclear transport are not cleaved after transport; NLS (nuclear localization sequence), because transport in the nucleus is required after each cell division RNA molecules are exported to the cytosol. Nuclear proteins (RNA & DNA polymerases, histones, topoisomerases) are synthesized in the cytosol and imported into the nucleus. The signal sequence that targets a protein to the nucleus (NLS) is not removed after the protein arrives at its destination. Several proteins that cycle between the nucleus & the cytosol mediate the process. Importin α,β and a small GTPase known as Ran. CAS (cellular apoptosis susceptibility protein).  Post-synthetic modification & incorporation Protein modification Outcome Phosophorylation (transient) Add a phosphate to serine, threonine & tyrosine Ubiquitination (transient) Add ubiquitin to lysine residue of a target protein for degradation Methylation (transient) Adds a methyl group usually at lysine or arginine residues Hydroxylation (permanent) Attaches a hydroxyl group to a side chain of a protein Glycosylation (permanent) Attaches a sugar usually to an N or an O in an a.a side chain Disulfide bond (permanent) Covalently links the S atoms of two different cysteine residues Acetylation (transient) Adds an acetyl group to the N terminus of a protein or the lysine residue Lipidation Attaches a lipid such as a fatty acid to a protein chain SUMOylation Add a small ubiquitin like modifier to a target protein  Protein degradation Prokaryotic: Involves chaperone-like proteins (Lon) Typically, ATP hydrolysis is used to manoeuvre a target protein through a pore into a proteolytic chamber , unfolding the protein in the process. Proteins are cleaved within the chamber. Eukaryotic: Ubiquitin is covalently linked to proteins slated for destruction via an ATP-dependent pathway that includes three separate types of enzymes: E1 activating, E2 conjugating and E3 ligases. Ubiquitinated proteins are degraded by a large complex known as the 26S proteasome. The 19S regulatory particle on each end of the core particle probably functions in unfolding the ubiquitinated proteins and translocating the unfolded polypeptide into the core for degradation  Protein-interacting molecule: ligand Binding site is complementary to ligand in size, shape, charge, hydrophobic or hydrophilic character The binding of the ligand induce a conformational change that makes the binding site more complementary (induced fit). The interaction between binding site and ligand is specific. Proteins are flexible and therefore changes in conformation can be subtle or more dramatic.  Protein interactions with different molecules Protein-protein interaction (antigen-antibody, enzymatic reactions, signalling, regulation) Protein-DNA interaction (Proteins involved in replication, transcription, translation) Protein-RNA interaction (proteins involved in translation and regulation of RNA stability) Protein interaction with other organic molecules (prosthetic groups, co-enzymes, signalling molecules) Protein interaction with anorganic molecules (co-factors) Principle of interaction: a mutual fit with respect to shape and chemical properties (hydrophilic/hydrophobic, polarity, charge  non-covalent interactions) Antigen binding to antibody (protein-protein interaction) The specificity is determined by the residues in the variable domains Induced fit: complementarity is achieved interactively as structures influence each other (occurs often) Antibody-binding site of antigen is called epitope Kd = 10^-10 M (strong interaction)  Protein interaction with nucleic acids (DNA, RNA) Basic amino acids (positively charged side chains interact with negatively charged phosphate groups in DNA/RNA backbone) Polar amino acid residues (interaction with nitrogen bases and sugar residues in DNA/RNA) involved in the interaction with DNA/ RNA Protein interaction with other organic molecules The active site is a 3D cleft formed by groups that come from different parts of the amino acid sequence, and it takes up a small part of the total volume of an enzyme. Substrates are bound by multiple (weak) interactions. The “flight or fight” response is common to many animals presented with a dangerous or exciting situation. The hormone epinephrine triggers the formation of cAMP which subsequently activates a key enzyme: protein kinase A (PKA). Protein interaction with anorganic molecules Carbon dioxide hydration and dehydration are often coupled to rapid processes catalysed by enzymes called carbonic anhydrases. The enzyme was found to contain a zinc ion which appeared to be necessary for catalytic activity In carbonic anhydrase three coordination sites are occupied by the imidazole rings of 3 His residues and an additional coordination site is occupied by a H2O molecule  Tertiary structure: Motifs & domains Motif (fold) is a recognizable folding pattern involving two or more elements of secondary structure and the connections between them. A motif can be very simple such as two elements of secondary structure folded against each other and represent only a small part of the protein (β-α-β loop). Domain is a part of a polypeptide chain that is independently stable or could undergo movements as a single entity with respect to the entire protein. In many cases a domain from a large protein will retain its native 3D structure even when separated from the remainder of the polypeptide chain. Different domains often have different functions, such as the binding of small molecules or interactions with other proteins  Same domains in different proteins proteins of protein families have similar amino-acid sequence and three-dimensional structure Example: Serine protease family Homeodomain is a DNA binding domain Most homeodomain proteins act as transcription factors & bind DNA to control the activity of other genes. In contrast to their similar DNA binding specificity, homeodomain proteins execute highly diverse & context- dependent functions. This, presumably, is why they are so highly conserved: they fulfil a function too vital to tolerate gross change in sequence or expression pattern. Yeast and Drosophila (fruit fly) are separated by more than a billion years of evolution but still have similar homeodomain sequences Conserved amino acid residues are important for protein function Conserved means the same in different organisms – these sequences did not change during evolution because they are important to the function of the protein Example IDH: Conserved residues implicated in the binding of Mg2+ (green), isocitrate (blue), and NADP (yellow); sequence comparisons can help to predict the function of yet unknown genes, e.g. containing NADP+ binding domain  Intrinsically disordered proteins Contain protein segments that lack definable structure (no domains) They lack a hydrophobic core and instead are characterized by high densities of charged amino acid residues such as Lys, Arg, and Glu. Disordered regions can conform to many different proteins, facilitating interaction with numerous different partner proteins Example: P53 protein with lots of ‘unstructured’ parts, binding to many interaction partners  Practice 1. What is a loop? Is the structure of a loop well defined? What can be a function of a loop? 2. What is meant with a motif and what with a domain? Can a motif be a domain? What is a subunit? What is meant with a protein family? What means ‘conserved’? 3. Which rest groups will have hydrophobic interaction (with each other)? And which H-bonds? And which ionic interactions?  Lesson 4: Protein motifs, domains, conserved regions Activation energy (EA or ΔG¥): energy required to initiate the conversion of reactants into products Catalyst provide an alternative route for the reaction! Enzymes are biocatalysts The active site is a 3D cleft formed by groups that come from different parts of the amino acid sequence. Substrates are bound to enzymes by multiple weak attractions (specificity) Noncovalent interactions between E and S (formation of ES complex). The interaction is mediated by the same type of interactions that stabilise protein structure. Formation of each weak interaction in the ES complex is accompanied by release of a small amount of free energy that stabilizes the interaction (binding energy ΔGB). This energy is used to lower the activation energy of reactions. Covalent interactions between enzyme and substrate; Catalytic functional groups on an enzyme may form a transient covalent bond with a substrate and activate it for reaction. These interactions lower the activation energy by providing an alternative (and of lower energy) reaction-path.  Enzymes are biocatalysts Enzymes facilitate the formation (stabilization) of the transition state. Enzymes accelerate reaction by lowering the activation energy Enzymes alter the rate of the reaction by they don't alter the equilibrium of a chemical reaction  In this figure the rate of product formation can be seen with or without enzyme. Note that the amount of product formed is the same whether or not the enzyme is present but, in the present example, the amount of product formed in seconds when the enzyme is present might take hours (or centuries) to form if the enzyme were absent.  Cyclophilin: catalyses the isomerization of peptide bonds from trans- to cis- form at proline residues and facilitates protein folding.  Enzyme classification Class name Type of reaction catalysed Oxidoreductases Transfer of electrons (hydride ions or H atoms) Transferases Group transfer reactions Hydrolases Hydrolysis reactions (transfer of functional groups to water) Lyases Cleavage of C-C, C-O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds Isomerases Transfer of groups within molecules to yield isomeric forms Ligases Formation of C-C, C-S, C-O, & C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor Synthetase Catalyse condensations that use ATP or other NTP as energy source Synthase Catalyse condensations that do not need ATP or other NTP as energy source Phosphorylases Add phosphate group to molecule; use Pi (Kinases use ATP) Phosphatase Remove phosphate using H2O Dehydrogenase Removes hydrogen/hydride (belongs to oxidoreductase) Protease Breakdown of proteins (belong to hydrolase) Nuclease Breakdown of nucleic acids (belong to hydrolase; DNase, RNase)  Some enzymes require co-enzymes Polypeptides (covalently linked -amino acids) + possibly: Catalytic principles Cofactors functional non-amino acid component Acid-Base catalysis: Proton exchange between metal ions or organic molecules enzyme (E) and substrate (S) Coenzymes Covalent catalysis: A group of the E becomes organic cofactors needed for enzyme activity covalently modified (e.g. covalent bond with S) NAD+ in lactate dehydrogenase Metal Ion catalysis: Metal ions facilitate Prosthetic groups chemical rearrangements or binding step. cofactors that are tight and permanently Catalysis by approximation: The enzyme holds attached 2 S near in space and in the correct orientation to heme in myoglobin optimize their reaction.  Acid-Base catalysis Negative charge attracts H+ Positive charge attracts electrons from water  OH- of water is This bond needs from O, away from C  N is more more reactive to attack C of to be cleaved prone to get attacked by water C=O  Covalent catalysis Rate acceleration by transient formation of a covalent Catalysis by enzyme substrate bond The reaction can’t go back because displaced group has been approximation released The enzyme alters pathway to get to the product by stabilizing Proximity: reaction between bound molecules the intermediate with the covalent bond, but if a covalent bond doesn't require an improbable collision of 2 is formed at an intermediate step the bond must be broken in a molecules. They are already in “contact” subsequent step to finish the reaction (increases local concentration of reactants) Orientation: reactants are not only near each Metal ion catalysis: carbonic anhydrase other on enzyme they are oriented in optimal position to react. The improbability of colliding in correct orientation is taken care of  Other effects that stabilize the transition state Electrostatic effects: increase in strength of ionic interactions due to lower dielectric constants Desolvation: exclusion of water from active site Induced fit: change of confirmation of enzyme or substrate to optimize interactions Enzyme kinetics Kinetics is the study of the rate at which compounds react Rate of enzymatic reaction is affected by: Enzyme Substrate Effectors Temperature  Lesson 5: Enzymes & enzyme kinetics The more S present, the higher the velocity The Vmax is reached when all enzymes' molecules are bound to S (evidence for the existence of ES). Catalyze reaction have a saturation effect  maximum velocity Reaction rate: the change in the [conc.] of a reactant or a product with time (M/s) Unimoleculat reaction A products rate= k[A] Rate is the velocity of the reaction (V): V= k[A]  Michaelis & Menten An E combines with S to form an ES complex with a rate constant k1. The ES complex has 2 possible fates: It can dissociate to E and S with a rate constant of k-1 or it can proceed to form a complex P with a rate constant k2. The latter reaction is slower therefore it is the one which determines the rate of the whole reaction series. The whole reaction series must be for this reason proportional to the concentration of the species that reacts in the second step, that is ES. We simplify these reactions by considering the rates of reaction at times close to zero. We also take for granted that [S] >> [E]: steady state assumption -> rate of ES formation = rate of ES breakdown  Relationship between Vmax, Km (& kcat) Michaelis-Menten equation: v =  rate of the reaction= velocity v– µmol L-1 min-1 = µM min-1 Vmax The higher the [S], the faster the reaction, until E is saturated with substrate: [ES] = [E]T (total enzyme conc.) Then enzyme concentration becomes the limiting factor Vmax is characteristic for each enzyme-substrate combination Vmax = k2[E]T Vmax, given in µmol L-1 min-1: Amount of substrate/product converted per volume per time  Enzyme kinetics: Kcat rate constant of a reaction k, given in units of reciprocal time (for first order reaction) Turnover number (kcat): number of S molecules converted into product by an enzyme molecule in a unit of time when the enzyme is fully saturated with the substrate. Vmax = k2[E]T = kcat [E]T → kcat =Vmax / [E]T kcat is the number of S molecules converted to P per time unit and E molecule when the E is saturated with S. kcat is rate limiting, thus kcat = 3 s-1 means: one enzyme molecule can convert 3 substrate molecules per second Km Km: Michelis-Menten constant – the substrate concentration at which V= Vmax/2 Under specific conditions: Km = [E][S] / [ES] Km is equal to the dissociation constant of the ES complex Km is a measure of the strength for the ES complex:  High Km indicates weak binding  Low Km indicates strong binding Unit for Km is mol/L (like S) Effective enzyme = High kcat, low Km & high kcat/Km  Lesson 6: In vivo regulation of enzymic activity Enzyme activity can be regulated by: regulation of the amount of enzyme that is expressed in the cell pH in tissue (digestive enzymes) The effect of pH on enzyme activity the rate of enzymatic reaction depends on pH of the medium each enzyme have pH where the enzyme is most active - which is known as the optimal pH (the range from pH5 to pH9) The optimal pH for an enzyme depends on where it normally works Extremely high or low pH values generally result in the complete loss of activity for most enzymes Enzyme activity can be regulated by: Allosteric modifications (allosteric modulators / effectors) Covalent modification (e.g. phosphorylation) Feedback control (cellular respiration) Proteolytic cleavage (digestive enzymes, zymogenes) Allosteric modifications The modulators for allosteric enzymes may be inhibitory or stimulatory. Often the modulator is the substrate itself. Regulation in which substrate and modulator are identical is referred to as homotropic (if different then heterotropic). Binding of the ligand or the substrate causes conformational changes that affect the subsequent activity of other sites on the protein. In most cases the conformational change converts a relatively inactive form (T state) to a more active conformation (R state) (cooperativity) Haemoglobin & O2 binding  Covalent modification Phosphorylation is a type of covalent modification that activates or deactivates an enzyme A kinase activates an inactive enzyme by phosphorylation A phosphatase activates an inactive enzyme by removal of a phosphate Another covalent modification would be the regulation of glycogen phosphorylase, which exist in 2 forms:  more active phosphorylase A  less active phosphorylase B Specific phosphorylation of less active phosphorylase B on Ser14 on each subunit by two molecules of ATP by phosphorylase kinase enzyme produced the more active phosphorylase A enzyme Similarly, the dephosphorylation by the enzyme phosphorylase phosphatase produce the less active phosphorylase B enzyme  Feedback control Feedback inhibition is a form of negative feedback by which metabolic pathways can be controlled In end-product inhibition, the final product in a series of reactions inhibits an enzyme from an earlier step in the sequence The product binds to an allosteric site and temporarily inactivates the enzyme (via non-competitive inhibition) As the enzyme can no longer function, the reaction sequence is halted, and the rate of product formation is decreased Feedback inhibition functions to ensure levels of an essential product are always tightly regulated If product levels build up, the product inhibits the reaction pathway and hence decreases the rate of further product formation If product levels drop, the reaction pathway will proceed unhindered, and the rate of product formation will increase  Proteolytic cleavage The inactive precursor is called a zymogen. Proteolytic activation in contrast with allosteric control and reversible covalent modification is irreversible. Some examples are digestive enzymes in the stomach and pancreas, blood clotting factors, protein hormones (e.g. proinsulin) Trypsin is the cleaved product of trypsinogen Enteropeptidase (Ser-protease) hydrolyses trypsinogen to trypsin Formed small amount of trypsin cleaves trypsinogen (autocatalytic)  Enzymic activity: Inhibition Major control mechanism in the biological systems Many drugs & toxic agents act as inhibitors Inhibition examining can be a source of insight for the mechanism for an enzymic reaction: Irreversible Reversible- competitive, Uncompetitive & Noncompetitive Irreversible inhibition: suicide inhibitors An irreversible inhibition dissociates very slowly from its target enzyme because it has become tightly bound to it, either covalently or non covalently Penicillin interferes with the synthesis of the bacterial cell wall (peptidoglycan) It forms a covalent bond with a Ser residue at the active site of the enzyme (transpeptidase) The transpeptidase is irreversibly inhibited and cell-wall synthesis cannot take place Reversible Inhibition: competitive Reversible inhibition is characterised by a rapid dissociation of the enzyme-inhibitor (EI) complex. In this type of inhibition, the E can bind the S or the I but not both. The inhibition can be overcome with excess of substrate. The competitive inhibitor often resembles the substrate. MTX is a competitive inhibitor of the enzyme dihydrofolate reductase (biosynthesis of purines and pyrimidines). Competitive Inhibitor binds, like the substrate, only at the active centre (competition) The hallmark of competitive inhibition is that it can be overcome by a sufficiently high [S]. The effect of a competitive I is to increase the apparent value of Km meaning that more S is needed to obtain the same reaction rate.  Reversible Inhibition: uncompetitive Uncompetitive inhibition is essentially substrate dependent inhibition in that the I binds only to the ES complex. Uncompetitive inhibition cannot be overcome by the addition of more substrate. The inhibitor binds only at the ES complex eg. lithium on inositol monophosphatase application as drug against manic depressive psychosis In uncompetitive inhibition the ESI does not go on to form any product. Because some unproductive ESI complex will always be present, Vmax will be lower in the presence of the inhibitor than in its absence. The uncompetitive inhibitor lowers the apparent value of Km because the inhibitor binds to ES to form ESI depleting ES. To maintain the equilibrium more S binds to E  Enzymic activity: Noncompetitive In noncompetitive inhibition the inhibitor and the substrate can bind simultaneously to an enzyme molecule at different binding sites. Unlike competitive inhibition, the I can bind to free E or the ES. A noncompetitive inhibitor acts by decreasing the concentration of functional enzyme than by diminishing the proportion of enzyme molecules that are bound to substrate. The net effect is to decrease the turnover number. Here inhibition cannot be overcome by increasing [S]. The inhibitor binds at a site distinct from the substrate active site, but it binds to either E or ES The inhibitor binds at a site distinct from the substrate active site, but it binds to either E or ES Why is Vmax lowered though the Km is unchanged? In essence the inhibitor simply lowers the concentration of functional enzyme. The resulting solution behaves as more dilute solution of enzyme.  Lesson 7: Protein function; structural proteins & globular proteins Fibrous proteins Polypeptide chains are folded into filaments or sheets (rod or thread shape chain) the fibrous proteins are water insoluble due to a high concentration of hydrophobic amino acid residues fibrous proteins are structural proteins usually play a protective or supporting role; e.g: collagen, keratin and elastin, they are usually used to construct connective tissue tendons bones and muscle fibers they have unique specific structure an amino acid exists to be functional Fibrous proteins: Collagen 3 separate polypeptides are super twisted about each other forming a right-handed super helical twist. Only Gly residues can be accommodated at the very tight junctions between the individual chains (Gly-X- Y) H-bonds within a strand are absent. Instead, the helix is stabilized by steric repulsion of the pyrrolidine rings of Pro & Hyp.  Fibrous proteins: Collagen (Hyp) Collagen synthesized in the absence of ascorbate is less stable than the normal protein. Hyp stabilizes the collagen triple helix by forming interstrand hydrogen bonds. The abnormal fibers formed by insufficiently hydroxylated collagen account for the symptoms of scurvy. Forces the proline ring into a favourable pucker Offer more hydrogen bonds between the three strands of collagen The post-translational processing is catalysed by prolyl 4-hydroxylase and requires α-ketoglutarate, molecular oxygen, & ascorbate (vitamin C) Fibrous proteins: α-Keratin The surfaces where the two a-helices touch are made up of hydrophobic amino acid residues, their R groups are meshed together in a regular interlocking pattern. That permits a close packing of the polypeptide chains within the left-handed supertwist. Two α-helices interwind with each other to form a super twisted coiled coil (rich in hydrophobic amino acids) The α-keratin helix is right-handed while the helical path of the supertwists is left-handed The tertiary structure of keratin is quite simple (left-handed super helix; coiled coils) The quaternary structure can the result of the assembly of coiled coils into supramolecular complexes The strength of fibrous proteins is enhanced by covalent cross-links between polypeptide chains. In α-keratins these are disulfide bonds.  Globular proteins Polypeptide chains tightly folded into compact spherical or globular shape most are soluble in water & biologically active Examples on globular proteins are: hemoglobin & myoglobin plasma proteins (proteins present in blood plasma), such as: albumin enzymes & protein hormones Haemoglobin oxygen needs to go from lungs to tissue oxygen is poorly soluble in aqueous solutions cannot be carried to tissues if simply dissolved in blood serum  transport mechanism necessary for multicellular organisms Haemoglobin in red blood cells transports oxygen Hemoglobin vs. myglobin 2 a-globin subunits 2 b-globin subunits Each subunit has one heme molecules (prosthetic group) Myoglobin exists as a single polypeptide whereas haemoglobin comprises four polypeptide chains that work cooperatively (the binding of an oxygen to a site in one chain increases the likelihood that the remaining chains will bind oxygen).  Heme Myglobin It gives their distinctive red colour Myoglobin is a single polypeptide (153 The organic component is called protoporphyrin amino acid res.) with one molecule of Only the Fe2+ (and not the Fe3+) can bind O2 heme It is located in the muscles Fe2+ can make 6 coordination bonds Facilitates the diffusion of O2 through the  4 with the nitrogen atoms of the ring cell for the generation of cellular energy  1 with the imidazole ring of a histidine residue  1 with O2 No cooperative binding possible High affinity for oxygen => Releasing O2 Protoporphyrin is made up of 4 pyrrole rings linked by methine only in low PO2 groups to form a tetrapyrrole ring. When O2 binds the electronic properties of iron change and this Myoglobin exists also in two forms: deoxy accounts in change of colour from the dark purple of oxygen myoglobin or oxymyoglobin depleted venous blood to the bright red of oxygen rich arterial blood. Thus, required properties are: Haemoglobin: O2 Efficient uptake of oxygen at high oxygen pressure (lungs) transportation Efficient release of oxygen at low oxygen pressure (tissue)  Haemoglobin: T & R state Haemoglobin exists in two conformations: T & R The T state is stabilized by greater number of ion pairs which lie at the α1β2 and α2β1 interface. The binding of O2 to a haemoglobin subunit triggers a change in conformation to the R state. In this process some of the ion pairs that stabilize the T state are broken and some new are formed. If a protein with high affinity (R state) was employed for O2 transport it would be 98% saturated in the lungs but would remain 91% saturated in the tissues and so only 7% of the sites would contribute to oxygen transport. If the protein bound oxygen with a sufficient low affinity (T state) to release it in the tissues, it would not pick much oxygen in the lungs. A sigmoid curve can be viewed as a hybrid curve reflecting a transition from a low-affinity to a high-affinity state  Binding of oxygen changes shape of unit & the shape of subunit affects the shapes of other subunits Cooperativity oxygen bound unit causes other subunits to become relaxed the rich become richer cooperative binding Cooperative binding: Concerted model All molecules exist either in the T state or in the R state (undergo transition simultaneously) At each level of oxygen loading, an equilibrium exists between the T and the R states The equilibrium shifts from T -> R when the molecule is loaded with O2 Thus, the binding curve for haemoglobin can be seen as a combination of the binding curves that would be observed if all the molecules remained in the T state or if all of the molecules remained in the R state. Cooperative binding: Sequential As oxygen molecules bind, the tetramers convert from the model T to the R state yielding the sigmoid binding curve so The binding of a molecule O2 in one subunit changes the important for efficient oxygen transport. conformation of this subunit The first change induces changes in neighbouring subunits  Concerted and sequential models represent idealized limiting cases, which real systems may approach but rarely that increase their affinity for the ligand attain  Haemoglobin & Allosterism Haemoglobin is an example of an allosteric protein An allosteric protein is one which the binding a ligand to one site affects the binding properties of another site on the same protein The proteins adapt other conformations induced by the binding of ligands referred to as modulators Modulators can be inhibitors or activators (Haemoglobin modulators: CO2, H+ and 2,3 BPG) Normal ligand = modulator  homotropic interaction Normal ligand ≠ modulator  heterotropic interaction Haemoglobin modulators: CO2 & H+ (Bohr effect) The CO2 produced by oxidation of fuels in mitochondria is hydrated to form H2CO3 The H2CO3 dissociates to HCO3- and H+ which leads to a drop of pH Haemoglobin transports about 40% of the total H+ and 15- 20% of the CO2 The rest of the H+ is absorbed by the bicarbonate buffer and the rest of CO2 as HCO3-  Haemoglobin modulators: 2,3- biphosphoglycerate (BPG) 2,3 BPG binds in the central cavity of the tetramer but only to T state On T- to R transition this pocket collapses and 2,3-BPG is released. This is a case of heterotropic allosteric inhibition of O2 binding to haemoglobin by 2,3-BPG The more BPG present, the less binding affinity for oxygen (little effect in lungs with high oxygen pressure, higher effect in tissues) Adaptation to high altitudes by regulating the [conc.] of 2,3-BPG At high altitude, pO2 is lower  Upregulation of BPG concentration in blood Lower affinity for oxygen Little effect on binding of oxygen in lungs (see curve) Considerable effect on release in tissues (see curve)  Fetal oxygen delivery The fetus haemoglobin needs to have a higher affinity for oxygen than that of his mother Fetus synthesizes γ- subunits instead of β-subunits of haemoglobin: a2γ2 haemoglobin a2g2 haemoglobin has lower affinity for BPG than a2b2 haemoglobin  higher affinity for oxygen This difference in oxygen affinity allows oxygen to be effectively transferred from maternal to fetal red blood cells. Haemoglobin: CO Haemoglobin: Sickle cell poisoning anaemia CO is a colourless and odourless gas that One mutation of a Glu to Val in both b-globin chains of binds to haemoglobin at the same site as haemoglobin β (mutated form: Haemoglobin S) oxygen but 200-fold more tightly forming a In the deoxy S due to extra hydrophobic interactions an aggregate complex named carboxyhaemoglobin. is formed (therefore only the T state aggregates). CO binding is a case of competitive When haemoglobin S is deoxygenated, it becomes insoluble & inhibition forms polymers that aggregate to into tubular fibers In addition, red cells from sickle cell patients are more adherent to the walls of blood vessels Results: painful swelling of the extremities & higher risk of stroke or bacterial infection & anaemia  Pratice 1. Draw a sigmoidal and a hyperbolic oxygen binding curve. Which of the two binding curves belongs to which oxygen binding molecules? 2. What are allosteric inhibitors? 3. a. What is the Bohr effect? b. What is the benefit of the Bohr effect? 4. What shows us that haemoglobin works co-operatively? 5. Where do we find BPG? What is the effect of BPG ? What is the influence of high altitude on BPG levels.