Fundamentals of Biochemistry Lecture Notes 1014NSC PDF

lOMoARcPSD|10208404 1014NSC Biochemistry Complete Lecture Notes Fundamentals of Biochemistry (Griffith University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Olivia David...

lOMoARcPSD|10208404 1014NSC Biochemistry Complete Lecture Notes Fundamentals of Biochemistry (Griffith University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Lecture Notes Week 1 – Water and Weak interactions Water 1. Water plays a central role in the chemistry of all life 2. Proteins, polysaccharides, nucleic acids, and membranes all assume their characteristic shapes in response to water 3. The chemical properties of water are related to the functions of biomolecules, entire cells, an organisms. Weak interactions in aqueous solutions 1. Hydrogen bonding 2. Ionic interactions 3. Hydrophobic interactions 4. van der Waals interactions Weak interactions are crucial to macromolecular structure and function The structure of H2O - Important properties of water arise from its angled shape. - Angle of 104.5º between two covalent bonds. bonding orbital - sp3. - Polar O-H bonds are due to uneven distribution of charge - The oxygen nucleus attracts electrons more strongly than does the hydrogen nucleus — the electrons are more often in the vicinity of the oxygen atom (2δ - ) than the hydrogen (δ +). - Angled arrangement of polar bonds creates a permanent dipole for a water molecule. Hydrogen bonding - Water molecules attract each other due to their polarity. - A hydrogen bond is formed when a partially positive hydrogen atom attracts the partially negative oxygen atom of a second water molecule. - Hydrogen bonds can form between electronegative atoms and a hydrogen attached to another electronegative atom. Hydrogen bonding between water molecules - A water molecule can form up to four hydrogen bonds. - In liquid water at room temperature and atmospheric pressure water molecules are: o disorganized (randon) o in continuous motion - Each molecules forms hydrogen bonds with an average of only 3.4 other molecules. Hydrogen bonding of ice - In ice, each water molecule forms the maximum of four hydrogen bonds, creating a regular crystal lattice. - This crystal lattice of ice makes it less dense than liquid water, and thus ice floats on liquid water. 1|Page Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Water as a solvent - Water is a good solvent for charged and polar substances: o Amino acids and peptides o Small alcohols o Carbohydrates - Water is a poor solvent for nonpolar substances: o Nonpolar gases o Aromatic moieties o Aliphatic chains Polar, non-polar and amphipathic biomolecules Examples of hydrogen bond acceptors and donors H-bond acceptor: electronegative atoms such as O or N with an ion pair of electrons H-bond donor: Hydrogen atoms covalently bonded to another electronegative atom Some biologically important hydrogen bonds - Source of unique properties of water - Structure and function of proteins - Structure and function of DNA - Structure and function of polysaccharides - Binding of substrates to enzymes - Binding of hormones to receptors - Matching of mRNA and tRNA 2|Page Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Some biologically important hydrogen bonds Examples of hydrogen bond acceptors and donors - Hydrogen bonds are strongest when the bonded molecules oriented to maximize electrostatic interaction, which occurs when the hydrogen atom and the two atoms that share it are in a straight line - that is, when the acceptor atom is in line with the covalent bond between the donor atom and H - holding two hydrogen bonded molecules or groups in a specific geometric arrangement. Ionic interactions - Ionic (Coulombic) interactions o electrostatic interactions between permanently charged species, or between the ion and a permanent dipole - Water as solvent. Water dissolves many crystalline salts by hydrating their component ions. The NaCl crystal lattice is disrupted as water molecules cluster about the Cl- and Na+ ions. The ionic charges are partially neutralized, and the electrostatic attractions necessary for lattice formation are weakened. Hydrophobic interactions - The biologically important gases CO2 , O2 , and N2 are nonpolar. Nonpolar gases are poorly soluble in water. The movement of molecules - from the disordered gas phase into aqueous solution constrains their motion and the motion of water molecules and therefore represents a decrease in entropy. 3|Page Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Effects of nonpolar molecules in water - Nonpolar Amphipathic compounds force energetically unfavourable changes in the structure of water. - Eg: Long-chain fatty acids have very hydrophobic alkyl chains, each of which is surrounded by a layer of highly ordered water molecules. - Release of ordered water favours formation of an enzyme- substrate complexes. While separate, both enzyme and substrate force neighbouring water molecules into an ordered shell. Binding of substrate to enzyme releases some of the ordered water, and the resulting increase in entropy provides a thermodynamic push toward formation of the enzyme- substrate complex. Van der Waals interactions - van der Waals interactions arise when two uncharged atoms are brought very close together, their surrounding electron clouds influence each other. Random variations in the positions of the electrons around one nucleus may create a transient electric dipole, which induce a transient, opposite electric dipole in the nearby atom. The two dipoles weakly attract each other, bringing the two nuclei together. Ionization of water, weak acids, and bases - Pure water is slightly ionized. - The ionization of water is expressed by an equilibrium constant. - The pH scale designates the H+ and OH- concentrations. - Weak acids and bases have characteristic dissociation constants. - Titration curves reveal the pKa of weak acids. 4|Page Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Ionization of water - Water is a neutral molecule with a very slight tendency to ionize. H2O ↔ H+ + OH- - Free protons (H+) don’t actually exist, rather they exist as hydronium ions, H3O+. - For simplicity, however, we often represent these ions as H+. H2O ↔ H+ + OH - The ionization (dissociation) of water is described by the expression H +¿ ¿ OH −¿ ¿ ¿ K =¿ - where K is the dissociation constant - Because of the undissociated [H2O] is much larger than the concentrations of the component ions, it can be considered constant (unchanging), and incorporated into K to yield an expression for the ionization of water Kw. Kw = [H+] [OH- ] - The value of Kw, the ionization constant of water is 10-14 at 25°C. - If the ionization constant of water is 10-14 then the concentration of H+ in solution is 10-7 mol/L with an equivalent concentration of OH- ions. H +¿ ¿ OH −¿ ¿ OH −¿ ¿ H +¿ ¿ ¿ K w =¿ - Since these values are very low and involve negative powers of 10, the pH scale can be used. H +¿ ¿ H +¿ ¿ pH=−log 10 ¿ - Eg: Human blood plasma has a [H+] of ~ 0.4 x 10-7 mol/L or 10-7.4 mol/L which gives a pH of 7.4 - The value where an equal amount of H+ and OHions are present is termed neutrality: at 25°C the pH of pure water is 7.0. At this temperature pH values below 7.0 are acidic and above pH 7.0 are alkaline. - Neutral solutions change with temperature, due to enhanced dissociation of water with increasing temperature. 5|Page Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Always remember that the pH scale is logarithmic, and not a linear one. Thus, a solution of pH 3.0 is not twice as acidic as a solution at pH 6.0 but 1000 times more acidic (ie: contains 1000 times more H+ ions). Proton movement between water molecules - The proton of a hydronium ion can jump rapidly from one water molecule to another - Therefore, the mobility of H+ and OH ions in solution are much higher than for other ions. - Proton jumping is the reason for acid-base reactions being among the fastest. Acid-base chemistry - Acid – a compound that acts as a proton donor in an aqueous solution. - Base – a compound that acts as a proton acceptor in an aqueous solution. - Conjugate pair – an acid together with its corresponding base. - By the above definitions, an acid-base reaction can be written as HA + H2O ↔ H3O+ + A - An acid (HA) reacts with a base (H2O) to form a conjugate base of the acid (A- ) and the conjugate acid (H3O+). - Eg: acetate ion (CH3COO- ) is the conjugate base of acetic acid (CH3COOH) and the ammonium ion (NH4 +) is the conjugate acid of NH3. - The acid-base reaction is usually abbreviated to HA ↔ H+ + A - The participation of water in the reaction is implied. An alternative expression for a basic solution is HB+ ↔ H+ + B - The strength of an acid is specified by its dissociation constant, or its efficiency as a proton donor. - The equilibrium constant for an acid-base reaction is expressed as a dissociation constant. H 3O+¿ ¿ A−¿ ¿ ¿ [ products] K= =¿ [reactants] - In dilute solutions, the water concentration is essentially constant 55.5 M. Therefore, the term [H2O] is customarily combined with the dissociation constant, to take the form. −¿ - For an acid +¿+ A¿ HA=H ¿ 6|Page Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC H +¿ ¿ A−¿ - At equilibrium ¿ ¿ Ka=K [ H 2 O ] =¿ - Thus, the stronger the acid the greater the value of Ka. - Because the acid dissociation constants, like [H+] values are cumbersome to work with, they are transformed into pK values by the formula. pK = -logK analogous to pH = -log [H+] = log 1 [H+] - Acids can be classified according to their relative strengths, that is, their ability to transfer a proton to water. Weak acids K < 1 strong acids K >> 1 - Virtually all the acid-base reactions in biological systems involve H3O+, (OH- ) and weak acids (and their conjugate bases). - The pH of a solution is determined by the relative concentration of acids and bases. The relationship between the pH of a solution and the concentrations of an acid and its conjugate base can easily be derived. - The Henderson-Hasselbach Equation A−¿ ¿ ¿ ¿ pH = pKa+ log ¿ - Relates the extent of ionisation of a weak acid (and base) to the pH of the solution. [conjugate base] pH = pKa+log [acid ] - This equation is of fundamental importance in preparing buffer solutions to control pH during biochemical reactions. - The ionisation characteristics of some of these compounds/groups actually controls the pH in cells and physiological fluids so pH varies only over a narrow range. - In Blood the pH stays in the range 7.0 – 7.8 - The concentration of the acid in the original solution can be calculated from the volume and concentration of NaOH added and a titration curve plotted. - At the midpoint of the titration, at which exactly 0.5 equivalent of NaOH has been added, [HA]=[A- ] and pH=pKa. 7|Page Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - The titration curves of these acids have the same shape, they are displaced along the pH axis because the three acids have different strengths. Acetic acid, with the highest Ka (lowest pKa) of the three, is the strongest (loses its proton most readily); it is already half dissociated at pH 4.76. Dihydrogen phosphate loses a proton less readily, being half dissociated at pH 6.86. Ammonium ion is the weakest acid of the three and does not become half dissociated until pH 9.25. - Buffer are mixtures of weak acids and their conjugate bases. - Buffers are aqueous systems that tend to resist changes in pH when small amounts of acids or base added. - A mixture of equal concentration of acetic acid and acetate ion, found at the midpoint of the titration curve is a buffer system. Buffering in biological solutions - Buffers are mixtures of weak acids and their conjugate bases. - A simple expression relates pH, pKa, and buffer concentration (Henderson-Hasselbalch equation). - Weak acids or bases buffer cells and tissues against pH changes. Week 3 – Amino Acids and Peptide bonds - Amino acid: α-amino-substituted carboxylic acids, the building blocks of proteins - Peptide bond: A substituted amide linkage between the α-amino group of one amino acid and the α-carboxylic group of another. Amino acids - All amino acids have a carboxyl and amino group bonded to an α-carbon. - The α-carbon also has a hydrogen and a side chain (R-group). - Each amino acid differs in the side chain which vary in charge, size, structure, water solubility and chemical properties. Chirality of amino acids - All amino acids with the exception of glycine are chiral and thus exist in L and D isomers. - Only L-enantiomers are found in proteins. - The formation of stable repeating substructures in proteins generally require their constituent amino acids to be of one stereochemical series. - Cells are able to specifically synthesize the L isomer of amino acids because the active site of enzymes is asymmetric, causing the reactions they catalyse to be stereospecific. Non-polar aliphatic amino acids - Aliphatic amino acids tend to be hydrophobic. - Become more hydrophobic as the side chain increases in length. - Hydrophobic amino acids are usually buried in proteins for protection against aqueous environments. 8|Page Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Tend to be clustered together within proteins, stabilizing structure by means of hydrophobic interactions. - Methionine is one of only 2 amino acids that contain sulfur. It contains a nonpolar thioether group in its side chain. Proline - Proline is a cyclic amino acid. Shares many of the properties of aliphatic amino acids. The rigid nature of proline makes the folding of proline into proteins difficult. Generally introduces a kink into polypeptide chain. Polar, uncharged amino acids - R groups of these amino acids are more soluble in water and more hydrophilic than the nonpolar amino acids. - Side chains contain functional groups that are able to form hydrogen bonds in water. - Polarity is due to the hydroxyl groups of serine and threonine, the sulfhydryl group of cysteine and the amide groups of asparagine and glutamine. - Asparagine and glutamine are the amide derivatives of aspartate and glutamate, respectively. Cysteine - The side chain can ionize at moderately high pH. - It can form a covalently linked dimeric amino acid called a cystine in which two cysteine residues are joined to form a disulfide bond. - Play a special role in structures by covalently links between different parts of a protein or different polypeptides. - Disulfide linkages are strongly hydrophobic (nonpolar). Lysine and Arginine 9|Page Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - The R groups carry positive charges and are strongly polar at pH 7.0. - They are very hydrophilic and usually found on the exterior of proteins. - Lysine has a secondary amino group on its aliphatic side chain. - Arginine has a positively charged guanidino group. Histidine - Histidine contains a imidazole group. - Is the only common amino acid having an ionizable with a pKa near neutrality. - In enzyme catalysed reactions a his residue facilitates the reaction by serving as a proton donor/acceptor. Negatively charged amino acids - R groups carry a net negative charge at pH 7.0. - Like the positively charged amino acids they are very hydrophilic. - Both aspartate and glutamate have a second carboxylic acid group. Aromatic amino acids - The R groups in this class are relatively nonpolar and participate in hydrophobic interactions. - The hydroxyl group of tyrosine is also important because of its ability to form H-bonds, and it is an important functional group in some enzymes. - Tyrosine and tryptophan are significantly more polar than phenylalanine because of the tyrosine hydroxyl group and the tryptophan indole ring. - Tryptophan, tyrosine and to a lesser extent phenylalanine absorb ultraviolet light. Modified amino acids - There are a number of modified amino acids which are formed from the common amino acids. - Examples: o 4-hydroxyproline (cell walls, collagen) o 5-hydroxylysine (collagen) o 6-N-methyllysine (myosin) o γ-carboxyglutamate (prothrombin) o selenocysteine 4-hydroxyproline - Example: collagen, a fibrous protein of connective tissue. 5-hydroxylysine - Example: collagen, a fibrous protein of connective tissue 6-N-methyllysine - Example: a constituent of myosin, a contractile protein of the muscle. γ-carboxyglutamate - Example: found in the blood clotting protein prothrombin and in certain Ca2+ binding proteins. 10 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Selenocysteine - Is introduced during protein synthesis rather than created through a postsynthetic modification. Other properties of amino acids - Amino acids are ionised in aqueous solutions. - At pH 7.0, the amino group is largely protonated (NH3+) and the carboxyl group largely deprotonated (COO-) forming a zwitterion. Amino acids titration curves - Two distinct stages corresponding to the deprotonation of carboxy group and the amino group. - At low pH glycine is fully protonated. - At the midpoint of this part of the titration (point of inflection) the pH = pKa (pK1). - As the titration continues another inflection point is reached. At this point removal of the first proton is complete. - The second half of the titration corresponds to the removal of the proton from the amino. - It gives a quantitative measure of the pKa of each of the ionizing groups. - There are two regions of buffering power, extending for approximately 1 pH unit either side of the pKa. - The relationship between net electric charge and the pH of the solution can also be derived from the titration curve. So at pH 5.97, glycine is present in its dipolar form, fully ionized but with no net electrical charge. This point is called the Isoelectric point or Isoelectric pH. - So at a pH < pI glycine has a net + charge & at a pH > pI glycine has a net – charge Ionization of amino acids - The side chains of some amino acids are also ionisible and each group has a specific pKa. - At pH > pKa the proton tends to be off. - At pH < pKa the proton tends to be on. - The average pKa for an amino acid is called the isoelectric point (pI). - As pH increases above the pI the net charge becomes negative. - As pH decreases below the pI the net charge becomes positive. - When the amino acids are incorporated into proteins only the charges on the side chains remain. Peptide bond - A peptide bond is formed when two amino acids are covalently linked between the amino group of one amino acid and the carboxyl group of another. The result is a dipeptide. Properties of peptide bonds - Peptide bonds are planar and prefer to be in the trans form. - The peptide bond exhibits a partial double bond character. - Oligo and polypeptides have unreacted amino group at one terminus (the amino N terminus) and an unreacted carboxyl group at the other end (carboxy C terminus). Diversity of proteins 11 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Definition: A macromolecule composed of one or more polypeptide chains, each with a characteristic sequence of amino acids linked by peptide bonds. - Proteins are the most abundant biological molecules, occurring in all cells and all parts of cells. - Range in size from relatively small peptides to huge polymers of molecular weights in the millions. - Proteins occur in great variety and exhibit enormous diversity in biological function. - Enzymes - Transport proteins - Nutrient & storage proteins - Contractile & motile proteins - Structural proteins - Defence proteins - Regulatory proteins - Specialized proteins Enzymes - Definition: A biomolecule, either protein or RNA, that catalyzes a specific chemical reaction. - Eg: - Triose phosphate isomerase - Serine proteases – trypsin & chymotrypsin Transport proteins - Myoglobin & Hemoglobin transport O2 the lungs to peripheral tissues. - Lipoproteins in blood plasma carry lipids to the liver and other tissues. - Cell membrane proteins transport molecules and ions across membranes. Nutrient and storage proteins - Plant seeds store nutrient proteins needed for embryonic growth. - Ovalbumin (egg white) and Caesin (milk) are examples of animal nutrient proteins. - Other proteins store non-nutrient molecules and ions (eg: ferritin). Contractile and motile proteins - Actin and Myosin are the key proteins involved in the movement of skeletal muscle. - Tubulin microtubules are componentsof flagella and cilia which can move and propel cells. Structural proteins - Collagen (see figure) is a component of cartilage and tendons. - Elastin is found in ligaments and is a fibrous protein that stretches in two dimensions. - Keratin makes up part of the fingernails and hair. - Silk fibres and spider webs contain fibroin. Defence proteins - Immunoglobulins are specialised proteins made in lymphocytes that recognise and neutralise foreign antigens (bacteria, viruses and proteins). - Fibinogen & thrombin are involved in blood clotting to prevent blood loss. - Snake venoms, bacterial toxins & toxin plant proteins all function to protection of the organism. 12 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Regulatory proteins - Insulin is a hormone that regulates sugar metabolism. - Repressors are proteins that regulate the transcription of specific genes Specialised proteins - There are any proteins which have specialised functions that are not easy to classify. - Antifreeze protein from Antarctic fish which prevent their blood from freezing. Week 4 – Protein Structure Net charge of a peptide Consider the peptide AEILKVG What is the net charge at pH 3.0, pH 8.0 & pH 12 ? NH3 +-A-E-I-L-K-V-G-COOH 1. Remember that every peptide has an N-terminus and a C-terminus. 2. Identify any amino acids that have a side chain that can be ionised, such as side chains with positively and negatively groups. In the case of the above peptide this is Lysine (K) and Glutamic acid (E). 3. 3. Consider the pKa of each of the ionisable groups. pH > pKa the proton tends to be off. pH < pKa the proton tends to be on. pH = pKa half of the molecules are protonated and half are deprotonated. 13 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Protein Structure and Function - Proteins are polypeptides of defined sequences and the amino acid sequence is know as the primary structure. - It is the primary structure of the protein that determines the properties of that protein. The 3-dimensional structure of proteins - Protein molecules fold into unique 3-dimentional structures or shapes based on their amino acid sequence. - The function of a protein is determined by its 3D structure. Structural organisation - Regions of polypeptide chains can fold into highly regular shapes and these folds are termed secondary structure. α-helix β-sheet - These secondary structure elements then fold into a unique 3-dimensional structure. This is called the tertiary structure. - When several units combine to form a protein containing several polypeptide chains, the level of organisation is called the quaternary structure. α-helices - α-helices are formed when the polypeptide strand folds around a central axis forming a regular, repeating helical structure. - Each repeating unit is a single turn which extends about 5.4 Å along the axis. - Each helical turn includes 3.6 residues. - The helical twist of α-helices in proteins are right-handed. - Naturally occurring L-amino acids can form either right- or left-handed α-helices. However, extended left-handed α-helices have not been observed in proteins. α-helices – stabilised by H-bonding - The amide H of each peptide linkage forms hydrogen bonds with the carbonyl O of the peptide bond 4 residue away, holding the helical structure together. - Every peptide bond of a α-helical polypeptide participates in H-bonding so that each successive turn of the α-helix is held to other turns by several H-bonds. This give the helical structure considerable stability. 14 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - The side chains of amino acids in a α-helix project outwards from the helical polypeptide backbone. α-helix stability - Not all polypeptides can form a stable α-helix. - Interactions between side chains can stabilize or destabilise α-helical structure. o If a polypeptide contains a sections of glu residues, the segment cannot form an α- helix (at pH 7) because the negatively charged carboxyl groups of adjacent Glu residues will repel each other. o The same is true of adjacent positively charged lys and/or arg residues. o The bulk and shape of Asn, Ser, Thr and Cys residues can also destabilize an α-helix if they are to close together. - The twist of an α-helix ensures that critical interactions occur between an amino acid side chain and a side chain three (or 4) residues away. o positively charged residues are usually found 3 residues away from negatively charged residues, permitting the formation of ion pairs. o Aromatic residues are similarly spaced, resulting in hydrophobic interactions. Amino acids incapable of forming α-helices - Proline: The nitrogen atom is part of a rigid ring and rotation about the N-Cα bond is not possible and results in kinks which destabilize the helix. - Glycine: Occurs infrequently in α-helices because it has more conformational flexibility than other residues and tends to take up coiled structures quite different from an α-helix. α-helical dipole - A small electric dipole exists in each peptide bond. These dipoles extend through the hydrogen bonds of the helix, resulting in a net dipole along the helix that increases in helix length. - The partial + and – charges of the helix dipole reside on the peptide amino and carbonyl groups near the amino- and carboxyl-terminal ends of the helix, respectively. Thus, negatively charged amino acids are found at the amino terminus where they have a stabilizing interaction with the positive charge of the helix dipole. On the other hand, positively charged residues at the amino terminus would be destabilizing. - The opposite is true for the carboxyl terminal end of the helical segment. Summary of constraints affecting stability of an α-helix 1. Electrostatic repulsion between successive charged residues. 2. Bulkiness of adjacent R-groups. 15 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC 3. Interactions between R groups spaced three (or four) residues apart. 4. The occurrence of Proline and Glycine residues. 5. The interaction between amino acid residues at the end of a helical segment due to the inherent electric dipole to the α-helix. β-pleated sheet - In the β-conformation, the backbone of the polypeptide chain is extended or stretched out into a zigzag structure. This is called a β-strand. - The zigzag polypeptide chains (β-strands) are arranged side by side to form a structure resembles a series of pleats, is called a β-sheet. - Interchain H-bonds between peptide linkages in adjacent polypeptide chains stabilise the β- sheets. - The individual segments that form a β-sheet are usually nearby in a polypeptide chain but can be quite distant in the linear amino acid sequence. - The R groups of adjacent amino acids protrude from the zigzag structure in opposite directions, creating an alternating pattern. β-sheets - β-sheets can be formed between parallel or anti-parallel - β-strands (having the same or opposite amino-tocarboxyl orientations). - The structures are similar, although the repeat period is shorter for the parallel conformation and the hydrogen bonding patterns are different. - When two or more β-sheets are layered close together within a protein, the R groups of residues on the touching surfaces must be relatively small. o Eg: The β-sheets of fibroin of silk and spider webs are rich in Gly and Ala residues, the two amino acids with the smallest R groups. β-turns - Connect two adjacent strands in antiparallel β-sheets. - It is a 180 degree turn involving 4 amino acid residues. - The carbonyl oxygen of 1st residue forms a H-bond with amide hydrogen of the 4th. - Glycine and proline are often occur in β-turns. - Generally found near the surface of a protein. Proline in β-turns - Cyclic amino acid. - Introduces a kink into a polypeptide chain. - 6% of proline residues are in the cis configuration. (99.95% of amino acids other than proline are in the trans configuration) - Many of these cis-prolines are found in β-turns Random coil - The loops and turns within a protein that join the secondary structures together are designated as random coil. - These are generally Irregular arrangement of the polypeptide chain. - Regions of some proteins are disordered but become ordered when those regions interact/bind with other proteins. 16 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Motif (or structural motif) - Any distinct folding pattern for elements of secondary structure observed in one or more proteins. - A motif can be simple or complex and represents a part of a polypeptide chain. - Also called a fold or super secondary structure. - Structure motifs may not retain their structure and function when separated from the protein. Domains - A domain is a compact, locally folded region of tertiary structure. - Domains are interconnected by the polypeptide strand that runs the length of the protein. - Small proteins usually contain only one domain, but multiple domains are especially common to larger globular proteins. Different domains often perform different functions. - Domains often have stable structures when isolated from the native protein (eg. by proteolytic cleavage) and are often units of function. - Domains are frequently defined as part of a polypeptide chain that can independently fold. However, this does not necessarily apply to all units that are regarded as domains. - Where a protein contains more than one domain they usually have different functions. - They are usually constructed from a section of polypeptide that contains 50-350 residues. - Domains appear to be modular units from which proteins are constructed - As well as having distinct functions, domains can also serve as moving parts of a protein. This usually arises from flexible linkages between the domains. - Limited flexibility between domains is often crucial to substrate binding, allosteric control and assembly of large structures. Week 5 – Protein folding and fibrous proteins – structure and function Protein folding - The folding pathway of large polypeptides are complicated. There are several plausible models. 1. The folding process is hierarchical. Local secondary structures form first, α-helices and β- sheets. This is followed by longer range interaction between 2 helices coming together to form a stable super secondary structure. The process continues until complete domains form and the entire polypeptide is folded. 2. In another model, folding is initiated by a spontaneous collapse of the polypeptide into a compact state, mediated by hydrophobic interactions between nonpolar residues. - In principle the process of protein folding probably incorporates elements of both models Factors determining secondary and tertiary structure 17 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Evidence indicates that most of the information for determining the three-dimensional of a protein is carried in the amino acid sequence of that protein. - A protein can be denatured by heat, extremes of pH, or other compounds such as alcohol and urea, until they are an unfolded chain of random coil. The loss of structure means a loss of function. - By returning the protein to the right physiological conditions it is possible for the protein to refold into its 3-dimensional structure and thus regain its function. - But not for all proteins Denaturation of proteins - Protein have evolved to function under particular cellular conditions. - When conditions differ this can cause structural changes in the protein. - A loss of structure sufficient to result in a loss of function is called denaturation. - The denatured state does not necessarily result in complete unfolding but rather a randomization of the structure. - The tertiary structure of a protein is determined by its amino acid sequence. - The denaturation of proteins has been shown to be reversible. - Certain proteins can be denatured by heat or extremes of pH are able to regain their native structure and function if returned to suitable conditions. This is called renaturation - Conditions that can cause denaturation of proteins include: 1. High temperature 2. Extremes of pH – electrostatic repulsion 3. Organic solvents – alcohols or acetones 4. Solutes – urea or guanidine hydrochloride 5. Detergents Circular dichroism - Determination of Secondary Structure: Circular Dichroism (CD) Analysis o CD measures the molar absorption difference  of left- and right-circularly polarized light:  = L – R. o Chromophores in the chiral environment produce characteristic signals. o CD signals from peptide bonds depend on the chain conformation. Ribonuclease refolding experiment - Ribonuclease is a small protein that contains eight cysteines linked via four disulfide bonds. - Urea in the presence of 2- mercaptoethanol fully denatures ribonuclease. - When urea and 2-mercaptoethanol are removed, the protein spontaneously refolds, and the correct disulfide bonds are reformed. - The sequence alone determines the native conformation. - The experiment is quite “simple” but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize Thermodynamics of protein folding - How does the protein fold into its unique tertiary structure? - The folding of a globular proteins is clearly a thermodynamically favoured process under physiological conditions. 18 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - This means that the overall free energy change on folding must be negative. This negative free energy change is achieved by a balance of thermodynamic factors. o Conformational entropy o Non-covalent interactions (charge-charge interactions, internal hydrogen bonding, van der Waals interactions) o The hydrophobic effect Free energy equation ∆G = ∆H - T∆S How can proteins fold so fast? - Proteins fold to the lowest-energy fold in the microsecond to second time scales. How can they find the right fold so fast? - It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest-energy one is found (Levinthal’s paradox). - Search for the minimum is not random because the direction toward the native structure is thermodynamically most favourable. Conformational entropy - The folding process which goes from a multitude of random coil conformations to a single folded structure involves a decrease of randomness and thus a decrease in entropy. - This change is called the conformational entropy of folding. This requires energy - As proteins fold, proteins are stabilised by non-covalent interactions which is energetically favourable. Enthalpy of protein folding - The favourable enthalpy contribution arises from intramolecular side group interactions. - Maximising the non-covalent interactions supports the protein adopting a lower energy state. - Non-covalent interactions o hydrogen bonds o electrostatic interactions o hydrophobic interactions o van der Waals forces Hydrogen bonds - Many amino acids are either good hydrogen bond donors or acceptors (serine and threonine). - The amide protons and carbonyls in the polypeptide backbone that are not involved in secondary structure can form hydrogen bonds with side chains. Charge-charge interactions - Interactions between positively and negatively charged residues. - Sometimes called salt bridges, ionic interactions or electrostatic interaction. - Eg: a lysine side chain amino group close to the carboxylic acid group of a glutamate side chain results in an electrostatic attractive forces acting between them at neutral pH. 19 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - At extremes of pH these salt bridges are broken and thus contribute to the protein denaturation. Van der Waals interactions - Weak noncovalent interactions between uncharged groups. - Occur when two uncharged atoms are brought very close together and their electron clouds influence each other, resulting in transient electric dipole in the nucleus of one atom. This induces a transient opposite dipole in the nearby atom. - Can stabilise the tertiary structure of proteins. - Each individual interaction is quite weak contributing only a small amount of structural stability. - However, the sum of many of these weak interactions results in powerful stabilisation force. - On the other hand, these forces are weak they can be easily broken and reformed. Hydrophobic effect - Amino acids with hydrophobic side chains (leucine, isoleucine, phenylalanine, etc..) in the polypeptide, are buried within the core of the protein away from the aqueous environment further stabilizing the protein structure. What is the hydrophobic effect? - When a polypeptide chain is in an unfolded form, hydrophobic residues in contact with water cause ordering of the water molecules around the structure. But when the chain folds and the hydrophobic residues are buried in the structure, the ordered water molecules regain their freedom. - Thus, internalizing hydrophobic groups via folding increases the randomness of the whole system, and therefore, yields an entropy increase. Entropy of folding - Protein folding involves going from a multitude of random coiled conformations to a single folded structure. - This involves a decrease in randomness and thus a decrease in entropy. The change is termed the conformational entropy of folding. - Free energy equation ∆G = ∆H - T∆S - Where S is the entropy (degree of randomness) and H is the enthalpy (heat change in a constant-pressure reaction). - So, in protein folding the change in ∆S is negative which makes a positive contribution to ∆G. In other words the conformational energy change works against protein folding. - However, ∆G must be negative for the reaction to be energetically favourable. 20 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Therefore, a large negative ∆H is required for protein folding to be favourable or some other increase in entropy of folding. Both of these occur. - The major source of negative ∆H is energetically favourable interactions between groups within the folded molecule. - These include noncovalent interactions such as hydrogen bonds, van der Waals forces, hydrophobic interactions and charge-charge interactions. - An increase in entropy (∆S) is due to the hydrophobic effect. - The stability of the folded structure of a globular protein depends on the interplay of three factors. 1. The unfavourable conformational entropy change, which favours random chains. 2. The favourable enthalpy contribution arising from intramolecular side group interactions. 3. The favourable entropy change arising from the burying of hydrophobic groups within the molecule Hydrophobic effect - Hydrophobic bonding has been used to describe protein stabilization by burying hydrophobic residues. - The stabilization is not primarily the result of hydrophobic bonding but rather the overall stabilization of the system by a entropy effect (hydrophobic effect). - Cytochrome C has very small negative ∆S and myoglobin has a positive ∆S and these values come primarily from the hydrophobic effect. Cooperative folding - The relatively small free energy (∆G) corresponding the the folding reaction is usually the difference between large ∆H and T∆S terms. - These large values arise because proteins tend to fold in a cooperative manner. - A partially folded mixture contains wholly unfolded and wholly folded molecules, with few intermediate structures. - Thus, folding interactions within a molecule form and break in concert. As a consequence of this cooperativity, the thermal denaturation of proteins occurs over a narrow temperature range. Protein stabilisation with disulphide bonds - Once folding has occurred, protein structures are further stabilized by disulfide bonds between cysteine residues. o Eg:Bovine pancreatic trypsin inhibitor (BPTI)  3 disulfide bridges in 58 residues  It is one of the most stable proteins known  It is quite inert to unfolding reagents such as urea  Only exhibits thermal denaturation below 100°C in very acid solutions. Assisted protein folding - Not all proteins fold spontaneously as they are synthesized. 21 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Folding is facilitated by the actions of specialized proteins called molecular chaperones. - Molecular chaperones interact with partially folded or improperly folded polypeptides. - Molecular chaperones include Hsp70 (DnaK), Hsp40 (DnaJ) and GrpE. - Another group of chaperones are called chaperoins. These include GroES and GroEL. - Protein disulfide isomerase (PDI) o Catalyzes the interchange or shuffling of disulfide bonds until the native configuration of disulfide bonds in achieved. o Also catalyzes the elimination of folding intermediates with inappropriate disulfide cross-links. - Peptide prolyl cis-trans isomerase (PPI) o Catalyzes the interconversion of the cis and trans isomers of proline peptides bonds. Misfolding of protein - The misfolding of proteins may cause a loss of the correct tertiary structure. This would result in a loss of function as it is the 3-dimensional structure that ultimately determines the function of a protein. - Eg: Prions - Creutzfeldt-Jakob’s, Mad cow disease. Summary – Protein folding - Protein folding pathways with discrete intermediate structures. - Protein denaturation and renaturation - Protein structures are stabilised by non-covalent interactions - Protein contain regions which are unstructured (random coil, loops and turns) - Thermodynamics of folding is a combination of the entropy and enthalpy providing free energy. o Conformational entropy (random unfolded polypeptides folding into one structure) o Hydrophobic effect (ordered water molecules becoming randomised as the protein folds) o Maximisation of non-covalent interactions (enthalpy) Week 6 – Globular Proteins – structure and function Globular Proteins - Globular proteins are folded into compact structures (unlike the fibrous proteins). - The polypeptide chain can contain locally folded regions of secondary structure (α-helices and β-sheets). - It can also contains regions of irregular folding. - The complete 3-dimensional structure is called the tertiary structure. - The type and extent of the secondary structure elements within a globular protein vary enormously between different proteins. - To achieve a compact structure the polypeptide chain must have turns/loops which link the secondary structural elements. 22 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - In a compact structure approx. 1/3 of the amino acid residues are in turns or loops. - β-turns, play important roles and Proline is important in turns. - Bends and turns are usually found on the surface of proteins. Globular proteins – irregularly structured regions - Many globular proteins often reveal many strangely contorted loops and folds in chains. - These are usually referred to as random coil. - But, each of these regions appears to have its own particular fold, subsequently forming a rigid structure. This has been confirmed by X-ray diffraction. - This is in contrast to true random coil which is very flexible. Random coil cannot be resolved by X-ray diffraction. Globular proteins – general properties - Hydrophobic residues are generally found buried in the “core” or interior of the protein. - Charged and polar residues tend to arrange themselves near the outside or surface on the protein where they can interact with H2O and other polar molecules. - Polar side chains which are buried in the protein usually are found H-bonded to another side chains or peptide bonds. Quaternary structure of proteins - Refers to the arrangement of protein subunits in 3- dimensional complexes. - Range from simple dimers to large complexes. - Subunits can be the same or different. - The forces that stabilise the interactions between proteins and protein subunits are the same as those that stabilise tertiary structures (noncovalent interactions). - Polypeptide chains associate in order to perform a variety of functions. - Many multi-subunit proteins have a regulatory function, and the binding of a ligand can alter the function. - Separate subunits can carry out separate reactions. - Metabolic pathways are organised by the association of a number of separate polypeptides, allowing efficient channelling of pathway intermediates from one enzyme to the next. - Coat proteins in viruses, form primarily structural complexes. - Other quaternary complexes include collagen, DNA polymerase III holoenzyme of E. coli and ribosomal complexes. Aspartate carbamoyl transferase (ATCase) - ATCase can bind ATP/CTP and ATP and CTP compete for the same site. - The activity of ATCase is regulated by the ratio of ATP and CTP. - Allosteric regulation involves changes in the quaternary structure. - A major rearrangement of subunits occurs in the T → R transition. Pyruvate dehydrogenase complex - Is a complex of three enzymes, pyruvate dehydrogenase (E1 ), dihydrolipoyl transacetylase (E2 ) and dihydrolipoyl dehydrogenase (E3 ), with multiple copies of each. - PDH complex in mammals is 50 nm in diameter and five times bigger than the entire ribosome. 23 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - The substrate is channelled via a swinging arm action of lipoyllysyl of E2. Quaternary structure homo-multimers - Copies of the same polypeptide chain associated non-covalently. - These complexes are usually symmetrical (but not always). - May enzymes occur as dimers, trimers, tetramers, pentamers, heximers, octamers, decamers, dodecamers. - In many cases this allows allosteric cooperativity that results in increased activity. Hetero-multimers - Consists of two or more different polypeptide chains. Sometimes slightly different versions of the same polypeptide can associate. - Eg: Hemoglobin o An α-chain and a β-chain come together to form a heterodimer. o Two copies of this then form the normal hemoglobin tetramer. o This is equivalent to a α-chain dimer associated with a β-chain dimer. Reversible binding of ligands is essential - Specificity of ligands and binding sites - Ligand binding is often coupled to conformational changes, sometimes quite dramatically (induced fit). - In multisubunit proteins, conformational changes in one subunit can affect the others (cooperativity). - Interactions can be regulated. Protein interactions with other molecules - Reversible, transient process of chemical equilibrium: A + B ↔AB - A molecule that binds to a protein is called a ligand. o typically, a small molecule - A region in the protein where the ligand binds is called the binding site. - Ligand binds via same noncovalent interactions that dictate protein structure (see Chapter 4). o allows the interactions to be transient Protein-ligand binding - Consider a process in which a ligand (L) binds reversibly to a site in a protein (P). - This interaction can be described quantitatively by the association rate constant ka or the dissociation rate constant kd. 24 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - After some time, the process will reach the equilibrium where the association and dissociation rates are equal. K a [ P ] ∙ [ L ] =K d [ PL] - The equilibrium composition is characterized by the equilibrium associationconstant Ka or the equilibrium dissociation constant, Kd. Binding: in terms of the bound fraction - In practice, we can often determine the fraction of occupied binding sites (θ). [ PL] K a= [ P ] ×[ L] - Substituting [PL] with Ka [L][P], we’ll eliminate [PL]. - Eliminating [P] and rearranging gives the result in terms of equilibrium association constant. - In terms of the more commonly used equilibrium dissociation constant: - The fraction of bound sites depends on the free ligand concentration and Kd. - Experimentally: o ligand concentration is known o Kd can be determined graphically or via least-squares regression Binding specificity: Lock and key model - Proteins typically have high specificity: only certain ligands bind. - High specificity can be explained by the complementary of the binding site and the ligand. - Complementary in: o size o shape o charge o hydrophobic/hydrophilic character - The “lock and key” model by Emil Fisher (1894) assumes that complementary surfaces are preformed. Binding specificity: Induced fit - Conformational changes may occur upon ligand binding (Daniel Koshland in 1958). o This adaptation is called the induced fit. o Induced fit allows for tighter binding of the ligand. o Induced fit allows for high affinity for different ligands. - Both the ligand and the protein can change their conformations. 25 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Oxygen binding proteins - Myoglobin and Hemoglobin are O2 binding proteins. - They are arguably the most studied and best understood proteins. - They were the first proteins for which 3-dimensional structures were determined. - These molecules illustrate almost every aspect of that most central to biological processes, the reversible binding of a ligand to protein. - Classic model to study protein structure and function. - Biological problems: o Protein side chains lack affinity for O2. o Some transition metals bind O2 well but would generate free radicals if free in solution. o Organometallic compounds such as heme are more suitable, but Fe2+ in free heme could be oxidized to Fe3+ (very reactive!). - Biological solution: o Capture the oxygen molecule with heme that is protein bound. Myoglobin (storage) and haemoglobin (transport) can bind oxygen via a protein-bound heme. Oxygen - Oxygen is poorly soluble in aqueous solutions. Thus, it cannot be carried to tissues in sufficient quantities if it is simply dissolved in serum. - Diffusion of oxygen through tissues is inefficient over distances of more than a few millimetres. - Multicellular organisms have evolved proteins to store and transport oxygen. - However, no amino acid is capable of reversibly binding oxygen molecules. - Transition metals (in this case Fe) are able to bind molecular oxygen but they are to reactive. Thus, Fe is incorporated into a protein-bound prosthetic group called Heme. Heme prosthetic group - Heme consists of a complex organic ring structure protoporphyrin, to which a single ferrous (Fe2+) is bound. - The iron atom has six coordination bonds, four to nitrogen atoms that are part of the flat porphyrin ring and two perpendicular to the ring. 26 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - The coordination nitrogen atoms help prevent the heme iron atom from being converted to ferric (Fe3+) state. - Iron in the Fe2+ state binds oxygen reversibly. - One of the two remaining coordination sites is occupied by the side chain nitrogen of a His residue. - The final site is the binding site for molecular oxygen. Binding of carbon monoxide - CO has similar size and shape to O2 ; it can fit to the same binding site. - CO binds heme over 20,000 times better than O2 because the carbon in CO has a filled lone electron pair that can be donated to vacant d-orbitals on the Fe2+. - The protein pocket decreases affinity for CO, but it still binds about 250 times better than oxygen. - CO is highly toxic, as it competes with oxygen. It blocks the function of myoglobin, haemoglobin, and mitochondrial cytochromes that are involved in oxidative phosphorylation. Myoglobin - Myoglobin (Mb, Mr 16,700) is a simple oxygen-binding protein found predominantly in the muscle of almost all mammals. - It is a transport protein that facilitates oxygen diffusion in muscles. - It is typical of a family of molecules called globin, all of which have very similar tertiary structures. - The polypeptide is made up of 8 α-helices (labelled A-H) connected by bends (AB, CD, EF and so forth, reflecting the α-helical segments they connect) Haemoglobin - Haemoglobin (Hb, Mr 64,500) is tetrameric protein containing four heme prosthetic groups, one associated with each polypeptide chain. - The adult protein contains 2 α-chains (141 residues) and two β-chains (146 residues). - Fewer than half of the amino acid residues in the polypeptide sequence of the α and β subunits are identical. However, the 3-dimensional structures of the two types of subunits are very similar. - The haemoglobin subunits are also very similar to that of myoglobin even though the three polypeptides are identical in only 27 positions. Quaternary structure of haemoglobin - The quaternary structure feature strong interactions between unlike subunits. - α1β1 (and α2β2) interface involves 30 residues and remains intact when the tetramer is disassembled with urea. - The α1β2 (and α2β1) interface involves 19 residues and the interactions are mostly hydrophobic although there are many hydrogen bonds and a couple of ion pairs. Myoglobin and haemoglobin similarities - Mb and Hb polypeptides fold into very similar tertiary structures. - The backbone consists of 8 α-helices separated by bends with helices E and F forming the sides of the hemebinding pocket. 27 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - The side chains of amino acids extend outward from the helices occupying nearly all the space between the folded loops, excluding water from its interior. - All but two polar residues found on the outer surface and most of the hydrophobic residues are in the interior. All the proline residues coincide with loop regions. - The heme group is located within a pocket lined with hydrophobic amino acid side chains. Heme binding pocket - Heme is held in the hydrophobic pocket by coordination of the iron with His93 (hisF8). - On the other side, iron is coordinated with a water molecule (deoxy form) or an oxygen molecule (oxy form). - Another Histidine, His64 (hisE7) helps hold the oxygen molecule in place. Binding of oxygen to myoglobin - Oxygen binds to myoglobin reversibly, this simple reversible binding can be described by an association constant Ka or a dissociation constant Kd. - For a monomeric protein such as myoglobin, the fraction of binding sites occupied by a ligand is a hyperbolic function of ligand concentration. Binding of oxygen to haemoglobin - Hemoglobin undergoes a structural change on O2 binding. - Two major conformations of hemoglobin, the R-state and T-state. - O2 binds to either state, but has significantly higher affinity for hemoglobin in the R-state (oxyhemoglobin). - In the absence of O2 the T-state is more stable and is thus the predominant conformation (deoxyhemoglobin). - T and R were originally denote tense and relaxed, respectively - The binding of O2 to hemoglobin subunit in the T-state triggers a change in conformation to the R-state. - When the protein undergoes this transition, the structures of the subunits change very little but the αβ subunit pairs slide past each other and rotate, narrowing the pocket between the β subunits. - In this process some of the ion pairs that stabilise T state are broken and some new ones are formed. Cooperative binding of oxygen - For hemoglobin to be an efficient transporter of O2 from the lungs to the tissues it must be able to bind oxygen in the lungs where the pO2 is 13.3 kPa and release it in the tissues where the pO2 is about 4 kPa. 28 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - The problem: A protein with high affinity for O2 in the lungs would not be able to release it in the tissue, and a protein with high affinity for O2 in the tissue would not bind much O2 in the lungs. - Hemoglobin solve this problem by undergoing a transition change from low affinity state (T state) to a high affinity state (R state). As a result, Hemoglobin has a S-shaped or sigmoid binding curve for oxygen. - O2 binding to individual subunits of hemoglobin can alter the affinity for O2 in adjacent subunits. - The first molecule of O2 that interacts with deoxyhemoglobin binds weakly because it binds to a subunit in the T state. - Its binding, however, leads to conformational changes that are communicated to adjacent subunits, making it easier for additional molecules of O2 to bind. In effect the T → R transition occurs more readily in the second subunit once O2 is bound to the first subunit. - The last (fourth) O2 molecule binds to a heme in a subunit that is already in the R state and hence it binds with much higher affinity. - Cooperative binding of a ligand to a multimeric protein such as the binding of O2 to haemoglobin is a form of allosteric binding. - The binding of one ligand affects the affinities of the remaining unfilled binding sites and O2 can be considered as both a ligand and a homotropic modulator. - There is only one O2 binding site per subunit, so the allosteric effect gives rise to cooperativity mediated by conformational changes transmitted from one subunit to another by subunit-subunit interactions. Effects of other ligands on haemoglobin - Cooperative binding and transport of oxygen is only part of the allosteric behaviour of hemoglobin. - Hemoglobin functions efficiently in oxygen transport as a result of allosteric transition between high and low affinity states. - These allosteric changes can also be promoted by carbon dioxide, protons and other substances such as bisphospho-glycerate. These are termed allosteric effectors. Response to pH change – The Bohr effects - A decrease in pH lowers the affinity of Hemoglobin to bind oxygen. Hb.4O2 + nH+ ↔ Hb.nH+ + 4O2 - Actively metabolizing tissues generates H+ in capillaries and H+ promotes the release of O2. And when venous blood recirculates oxygenation releases H+. This tends to release CO2 from bicarbonate dissolved in the blood and CO2 is exhaled. - Mechanism – can be explained by the model of cooperative binding of O2. - H+ binds at higher affinity to deoxyHb. - In the deoxy form His146 forms a salt bridge with Asp94 (if His is protonated). - As a result of the salt bridge, His146 has a high pKa (6.5). - At pH 7.4, His 146 is largely unprotonated in the oxyHb form. - So, H+ favours the deoxyHb form and promotes the release of O2. Carbon dioxide transport - Release of CO2 from respiring tissue also lowers the O2 binding affinity of hemoglobin. 29 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Some CO2 becomes bicarbonate releasing H+ and contributing to the Bohr effect. - Some bicarbonate transported out of the erythrocytes is carried in the blood but a portion reacts directly with hemoglobin, binding to N-terminal groups of chains to form carbamates. -NH3 + + HCO3 - ↔ -NH-COO- + H+ + H2O - This carbamation reaction allows hemoglobin to aid in the transport of CO2 from tissues to lungs. - H+ is released on binding of HCO3 - and contributes to the Bohr effect. - A negatively charged group is introduced at the Nterminus of the chains stabilizing a salt bridge between the α and β chains, and this is characteristic of the deoxyHb state. - In tissue both the latter effect and lowering of the pH promote the release of O2 when CO2 is present. - The reverse reaction occurs in the lungs Summary of effects of H+ and CO2 - In the lungs where O2 is abundant, O2 binding favours the oxyHb form and CO2 is released. - In the tissues, CO2 is abundant, the pH is lowered by H+ and the deoxyHb form is favoured promoting the release of O2. 2,3-bisphosphoglycerate - BPG acts as an allosteric effector. - Binding of BPG acts to lower the oxygen affinity to haemoglobin. - BPG binds in the cavity between the β chains, making electrostatic interactions with positively charged residues around the opening. - BPG can only bind the deoxyHb form, because the cavity in the oxyHb form is to small to accommodate BPG. - The higher the BPG in red blood cells the more stable deoxyHb and a decrease in O2 affinity. Sickle-cell anaemia - Is a genetic disorder caused by a single amino acid substitution (Glu6 to Val6) in each β-chain of haemoglobin. The change introduces a hydrophobic patch on the surface of haemoglobin that causes molecules to aggregate into bundles of fibres. - Is a genetic disorder caused by a single amino acid substitution (Glu6 to Val6) in each β-chain of hemoglobin. The change introduces a hydrophobic patch on the surface of hemoglobin that causes molecules to aggregate into bundles of fibres. Week 7 – Enzymes - Physiological significance of enzymes - Origin of catalytic power of enzymes - Chemical mechanisms of catalysis - Mechanisms of chymotrypsin and lysozyme 30 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Description of enzyme kinetics and inhibition What are enzymes - Enzymes are catalysts. o increase reaction rates without being used up in the reaction. - Most enzymes are globular proteins. o However, some RNA (ribozymes and ribosomal RNA) also catalyse reactions. - The study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s. - The study of enzymes has dominated biochemistry in the past and continues to do so. Enzymes – definitions - Enzyme – A biomolecule, either protein or RNA that catalyses a specific chemical reaction. It does not affect the equilibrium of the catalysed reaction; it enhances the rate of the reaction by providing a reaction path with a lower activation energy. - Holoenzyme – A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions. - Apoenzyme/apoprotein – The protein part of an enzyme. - Cofactor – An inorganic ion or coenzyme required for enzyme activity. - Coenzyme – An organic cofactor required for the action of certain enzymes; often has a vitamin component. - Active site – the pocket on the enzyme where the reaction takes place. - Substrate – the molecule that is bound in the active site and acted upon by the enzyme Why biocatalysts over inorganic catalysts? 1. Greater reaction specificity: avoids side products 2. Milder reaction conditions: conducive to conditions in cells (Eg. pH ~ 7.0, 37°C) 3. Higher reaction rates: in a biologically useful timeframe 4. Capacity for regulation: control of biological pathways Enzyme – substrate complex drives selectivity E.g. Chymotrpsin - Binding of a substrate to an enzyme at the active site. - Key active-site amino acid residues on the surface are shown in red Enzyme – metal ion cofactors 31 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Enzyme – coenzymes Classification of enzymes Enzymes are divided into 6 classes based on the type of reaction catalysed Enzymatic catalysis – why are enzymes required? - Enzyme catalysis of reactions is essential to living systems o Under biological relevant condition, uncatalyzed reactions tend to be slow. o Biological molecules are stable in aqueous environment inside a cell (neutral pH & mild temperature). o Many common chemical processes are unfavourable in the cellular environment.  the transient formation of unstable charged intermediates  the collision of two or more molecules in the precise orientation required for the reaction - Enzymes circumvent these problems by providing a specific environment in which a given reaction can occur more rapidly. Enzymes enhance reaction rates 32 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC Enzymes - Enzyme as catalyst - Thermodynamics of enzyme catalyzed reactions - Types of catalysis o Acid-base catalysis o Covalent catalysis o Metal-ion catalysis - Enzyme specificity - Catalytic mechanisms - Enzyme examples o Chymotrypsin → mechanism & pH profile o Hexokinase → induced fit model o Enolase → catalysis involving a metal ion o Lysozyme → mechanism & pH profile Enzymatic catalysis - A simple enzymatic reaction might be written where E, S and P represent the enzyme substrate and product, ES and EP are transient complexes of the enzyme with the substrate and product. E + S ↔ ES ↔ EP ↔ E + P - The energy in a biological system is described in terms of free energy, G. - In a coordinate reaction diagram the free energy is plotted against the progress of the reaction. - The starting point for the forward or reverse reaction is called the ground state, the contribution to the free energy of the system by an average molecule (S or P) under a given set of circumstances. Enzymatic catalysis 33 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - The equilibrium between S and P reflects the difference in the free energies of their ground states. The ground state of P is lower than that of S, so ∆G ˊ˚ for the reaction is negative (exergonic) and at equilibrium there is more P than S (the equilibrium favours P). - The position and direction of equilibrium are not affected by any catalyst. - A favourable equilibrium does not mean that the S→P conversion will occur at a detectable rate. - The rate of the reaction is dependent on the energy barrier between S and P. o alignment of reacting groups o formation of transient unstable charges o bond rearrangements o other transformations required for the reaction to proceed - For the reaction to proceed, molecules must overcome this energy barrier, the at point decay to the S and P is equally probable. This is called the transition state. - The transition state is not a stable chemical species. - The difference between the energy levels of the ground state and the transition state is called the activation energy ∆G‡. - The rate of the reaction is reflected in the activation energy where the higher the activation energy the slower the reaction. - Reaction rates can be increased by: o raising the temperature o raising the pressure - Alternatively, the activation energy can be lowered by adding a catalyst. - Catalysts enhance the reaction rate by lowering activation energies. Enzymatic catalysis 34 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Enzymes are catalysts and do not affect the reaction equilibrium. - A simple enzymatic reaction might be written where E, S and P represent the enzyme substrate and product, ES and EP are transient complexes of the enzyme with the substrate and product. E + S ↔ ES ↔ EP ↔ E + P - The role of an enzyme is to accelerate the interconversion of S and P. - The enzyme is not used up in the process, and the equilibrium is unaffected. However, the reaction reaches equilibrium much faster in the presence of the enzyme because the reaction rate is increased. - Any reaction may have several steps, involving the formation and decay of transient chemical species called reaction intermediates. - When the S ↔ P reaction is catalysed by an enzyme, ES and EP complexes are considered intermediates. The ES and EP complexes occupy valleys in the reaction coordinate diagram. - Less stable chemical intermediates often exist in the course of an enzyme catalysed reactions. The interconversion of two sequential reaction intermediates thus constitutes a reaction step. - When several steps occur in a reaction, the overall rate is determined by the step (or steps) with the highest activation energy which is called the rate limiting step. The rate limiting step is the highest energy point in the diagram. - In practice the rate-limiting step can vary with reaction conditions, and for many enzymes several steps may have similar activation energies and therefore all partially rate-limiting. Understanding catalysis - Important to understand the distinction between reaction equilibria and reaction rates o The function of a catalyst is to increase the rate of the reaction. o Catalysts do not affect reaction equilibria - Note: A reaction is at equilibrium when there is no net change in the concentrations of reactants or products. - Any reaction, such as S ↔ P, can be described by a reaction coordinate diagram, a picture of the energy changes during a reaction Enzymatic reactions - Principle of the requirement for enzyme catalysed reactions - Eg: the conversion of sucrose and oxygen to carbon dioxide and water C12H22O11 + 12O2 ↔ 12CO2 + 11H2O - This conversion takes place via a series of separate reactions, and, has a very large and negative activation energy. At equilibrium, the amount of sucrose present is negligible. - However, sucrose is a stable compound and can be exposed to oxygen without reaction and therefore has a quite high activation energy barrier. - In cells, sucrose is readily broken down to CO2 and H2O in a series of enzyme catalyzed reactions. - The enzymes not only accelerate the reactions, they oraganise and control them so that the energy released is recovered in other chemical forms and redirected to other cell processes. Thermodynamics of reaction rates and equilibria 35 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Reaction equilibria are linked to the standard free energy change, ΔG ˊ˚, and the activation energy, ΔG‡. - An equilibrium reaction such as S↔P is described by the equilibrium constant, Keq (or simply K). Under standard conditions to compare biochemical processes is K ˊeq (K ˊ). Kˊeq = [P]/[S] - From thermodynamics the relationship between Kˊeq and ΔG ˊ˚ can be described by the expression. ΔGˊ˚ = -RT lnKˊeq T – absolute temperature 298 K (25˚C) R – gas constant 8.315 J/mol.K - A large negative value for ΔGˊ˚ reflects a favourable reaction equilibrium but this does not mean the reaction will proceed at a rapid rate. - The rate of any reaction is determined by the concentration of the reactants and by the rate constant, k. For a unimolecular reaction such as S→P, the rate (velocity) of the reaction, V represents the amount of S that reacts per unit of time expressed by the rate equation. V=k[S] Enzymatic catalysis - Enzymes do not affect equilibrium (Keq) - which means that enzymes cannot affect the free energy of the reaction (ΔG). - Slow reactions face significant activation barriers (ΔG‡) that must be surmounted during the reaction. - Enzymes increase reaction rates (k) by decreasing ΔG‡. - Transition state theory relates the magnitude of the rate constant to the activation energy. (k B T ) −∆ G‡ k= exp ⁡( ) (h) RT kB - Boltzman’s constant 1.381 x 10-23 J/K h – Planck’s constant 6.026 x 10-34 J.s T – absolute temperature 298 K (25˚C) R – gas constant 8.315 J/mol.K - Enzymes are exceptional catalysts for two main reasons: 1. Catalytic power – enhancing reaction rates (5-17 orders of magnitude) 2. Specificity – very specific, discriminating between substrates with quite similar structure - Both are related to protein stability provided through non-covalent interactions. Enzymatic catalysis – catalytic power - The catalytic power of enzymes is derived from the free energy released in forming many weak interaction between an enzyme and its substrate. - This binding energy contributes to the specificity as well as catalysis. Enzymatic catalysis – specificity 36 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Weak interactions are optimised in the reaction transition state; enzyme active sites are not complementary to their substrates per se but to the transition states through which substrates pass as they are converted to products during the enzymatic reaction. - Weak interactions are optimised in the reaction transition state. - The lock and key model the specific interaction between enzyme and substrate, however this can be misleading with enzymes as their active sites generally complementary to the transition state. Enzymatic catalysis - Real enzymes form weak interaction in the ES complex but the full complement of interactions between the enzyme ad substrate are only formed when the substrate reaches the transition state. - The free energy (binding energy) released by the formation of these interactions partially offsets the energy required to reach the top of the energy hill. - The summation of the unfavourable (positive) activation energy (ΔG‡) and the favourable (negative) binding energy (ΔGB ) results in a lower net activation energy. - In an enzyme reaction the transition state is a stable species but for a brief period of time that the substrate spends at the top of the energy hill. - The important principle is that weak interactions between the enzyme and substrate provide a substantial driving force for enzyme catalysis. - The physical and thermodynamic factors contributing to ΔG‡, the barrier to a reaction might include: 1. The entropy of molecules in solution, which reduced the possibility that they will react together. 2. The solvation shell of hydrogen-bonded water that surrounds and helps to stabilize most biological molecules in aqueous solution. 3. The distortion of substrates that must occur in many reactions. 4. The need for proper alignment of catalytic functional groups on the enzyme - Note: the binding energy can be used to overcome all these barriers. Enzymes – rate enhancement by entropy reduction - To enhance a reaction, a large restriction in the relative motions of two substrates that are to react is required. This would cause a reduction in the entropy but has a significant benefit to binding the substrates to the enzyme. - Binding energy constrains the substrates in the orientation required for the reaction to proceed and have a substantial contribution to catalysis, because productive collisions between molecules in solution can be exceedingly rare. - Substrates can be precisely aligned on the enzyme, with many weak interactions between each substrate and strategically located chemical groups on the enzyme clamping the substrate molecules in the proper positions. Enzymes – desolvation - The formation of weak bonds between substrate and enzyme results in desolvation of the substrate. - Enzyme-substrate interactions replace most or all the hydrogen bonds between the substrate and water that would otherwise impede the chemical reaction. Enzymes – weak interactions and alignment of functional groups 37 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Binding energies involving weak interactions formed only in the reaction transition state helps compensate thermodynamically for the unfavourable free energy change associated with any distortion, primarily electron redistribution, that the substrate must undergo to react. - The enzyme itself undergoes a change in conformation when the substrate binds, induced by multiple weak interactions with the substrate. This is referred to as induced fit. - The motions can affect a small part of the enzyme near the active site of can involve a large changes to bring specific functional groups on the enzyme into the proper positions to catalyse the reaction. Specific catalytic groups contribute to catalysis - Once a substrate is bound to enzyme, properly positioned catalytic functional groups aid in the cleavage and formation of bonds through a variety of mechanisms. o Acid-base catalysis o Covalent catalysis o Metal ion catalysis General acid-base catalysis - Acid-base catalysis involving the transfer of a proton is the single most common reaction in biochemistry. - One or many proton transfers occur in the course of most chemical reactions that take place in the cell. - General acid-base catalysis becomes crucial in the active site of an enzyme where water may not be available as a proton donors or acceptors. Several amino acids can take on the role of proton donors or acceptors. - These groups are positioned precisely in the active site tom allow proton transfers, with reaction rate enhancements of the order of 10 2-105. Covalent catalysis - Covalent catalysis involves a transient covalent bond being formed between the enzyme and the substrate. - Eg: Hydrolysis of a bond between A and B. - In the presence of a covalent catalyst (an enzyme with a nucleophilic group X:) the reaction becomes - The formation and breakdown of a covalent intermediate creates a new pathway for the reaction, but catalysis will only occur if the new pathway has a lower activation energy than the uncatalyzed pathway. - Both of the new steps must be faster than the uncatalyzed reaction Metal ion catalysis 38 | P a g e Downloaded by Olivia Davidson ([email protected])  lOMoARcPSD|10208404 Fundamentals of Biochemistry Trimester 2, 2021 1014NSC - Metal ions either bound to the enzyme or added in association with a substrate can participate in catalysis in many ways. o Ionic interactions between the enzyme-bound metal and substrate can help orientate the substrate for the reaction or stabilise charged reaction transition states. o Metal ions can mediate oxidation-reduction reactions by reversible changes in the metal ion’s oxidation state. - Approximately one third of all know enzymes require one or more metal ions for catalytic activity. Enzyme reaction mechanisms - To understand the complete mechanism of action of an enzyme we need to identify all substrates, cofactors, products and regulators, as well as: 1. The temporal sequence in which enzyme-bound reaction intermediates form. 2. The structure of each intermediate and each transition state. 3. The rates of interconversion between intermediates. 4. The structural relationship of the enzyme to each intermediate. 5. The energy contribution of all reacting and interacting groups to the intermediate complexes and transition states. - There are very few enzymes for which all this information is known. Nucleophiles and electrophiles - Nucleophile – An electron rich group with a strong tendency to donate electrons to an electron deficient nucleus (electrophile); the entering reactant in a biomolecular substitution reaction. - Electrophile – An elec