Biochemistry Lecture Slides PDF

BIOC 460/660 Foundations of Biochemistry and Molecular Biology I Lecture Slides - Qtr 1 Chapter 1.1-1.5 The Foundations of Biochemistry Chapter 13.1-13.2 Introduction to Metabolism Chapter 2.1-2.3 Water, the Solvent of Life Chapter 3.1-3.4 Amino Acids, Peptides, and Proteins Point...

BIOC 460/660 Foundations of Biochemistry and Molecular Biology I Lecture Slides - Qtr 1 Chapter 1.1-1.5 The Foundations of Biochemistry Chapter 13.1-13.2 Introduction to Metabolism Chapter 2.1-2.3 Water, the Solvent of Life Chapter 3.1-3.4 Amino Acids, Peptides, and Proteins PointSolutions Session: fabioc460 Chapter 1 The Foundations of Biochemistry © 2021 Macmillan Learning Chapter 1: The foundations of biochemistry Learning goals: ▪ Distinguishing features of living organisms ▪ Structure and function of the parts of the cell ▪ Roles of small and large biomolecules ▪ Energy transformation in living organisms ▪ Regulation of metabolism and catalysis ▪ Coding of genetic information in DNA ▪ Role of mutations and selection in evolution Biochemistry is the chemistry of living matter Living matter is characterized by: ▪ A high degree of complexity and organization ▪ The extraction, transformation, and systematic use of energy to create and maintain structures and to do work ▪ The interactions of individual components being dynamic and coordinated ▪ The ability to sense and respond to changes in surroundings ▪ A capacity for fairly precise self-replication while allowing enough change for evolution Three distinct domains of life: Cellular and molecular evolution Phylogenetic relationships illustrated by “family tree” of sorts Basis for relationship is often the similarity in ribosomal RNA (rRNA) sequences The more closely related two sequences are, the closer the “branches” The cell: The universal building block Living organisms are made of cells The simplest living organisms are unicellular (single-celled) Some simple organisms (bacteria/archaea) can be aerobic (derive energy from electron transfer to oxygen) or anaerobic (derive energy by electron transfer to nitrate, sulfate, or carbon dioxide) Larger organisms are multicellular (many-celled), with different functions for different cells Cells have some common features but can contain unique components for different organisms  Kingdom Cellular Organization Kingdoms: Defined by organism, Archaea → Unicellular prokaryote cellular, and molecular differences Bacteria → Unicellular prokaryote All cells share some common structures: Protista → Unicellular eukaryote ▪ Plasma membrane Fungi → Uni- or multicellular eukaryote ▪ Cytoplasm Plantae → Multicellular eukaryote ▪ Ribosomes Animalia → Multicellular eukaryote ▪ Genetic material Some cells are more complex…also contain: ▪ Membrane-bound organelles  Bacteria are the simplest of cells (mostly Bacterial cell structure made up of basic cellular features) Can also have some additional unique features, such as pili and flagella Thought to be the ancestor to the eukaryotic mitochondion Structure Composition Function Cell wall Carbohydrate + protein Mechanical support Cell membrane Lipid + protein Permeability barrier Cytoplasm Aqueous solution Site of metabolism Ribosomes RNA + protein Protein synthesis Nucleoid DNA + protein Genetic information Pili Protein Adhesion, conjugation Flagella Protein Motility Eukaryotic cells: More complexity Have membrane-bound nucleus by definition: ▪ Protection for DNA; site of DNA metabolism ▪ Selective import and export via nuclear membrane pores Have membrane-enclosed organelles: ▪ Mitochondria for energy in animals, plants, and fungi ▪ Chloroplasts for energy in plant ▪ Lysosomes for digestion of un-needed molecules Compartmental segregation of energy-yielding and energy-consuming reactions helps cells to maintain dynamic homeostasis and stay away from equilibrium. Animal and plant cells contain identical AND unique components Cytoplasm Cytoplasm is a highly viscous solution where many reactions take place. ▪ The cytosol is very crowded (see right) Cellular organization is dynamic Cytoskeleton consists of microtubules, actin filaments, and intermediate filaments. ▪ Cellular shape and division ▪ Intracellular organization ▪ Intracellular transport paths ▪ Cellular mobility Shown at right are: ▪ Microtubules (green) connected to chromosomes (blue) via kinetochores (yellow) and pulling them to opposite poles, denoted by centrosomes (magenta) ▪ Intermediate filaments composed of keratin (red) are important for the structure of the cell itself Biochemistry is the chemistry of living matter The basis of all life is the chemical reactions that take place within the cell. Chemistry allows for: ▪ A high degree of complexity and organization ▪ The extraction, transformation, and systematic use of energy to create and maintain structures and to do work ▪ The interactions of individual components to be dynamic and coordinated ▪ The ability to sense and respond to changes in surrounding ▪ A capacity for fairly precise self-replication while allowing enough change for evolution Organisms can be classified by energy and carbon sources used The sun IS the reason there is life on Earth Only plants and few bacteria can utilize this vast energy source (phototrophs) ▪ Produce glucose via photosynthesis Animals require a chemical energy source (chemotrophs) ▪ Since chemical energy required is an organic compound (like glucose), we are chemoheterotrophs Living systems extract energy Energy input is needed in order to maintain life. From sunlight ▪ Plants ▪ Green bacteria ▪ Cyanobacteria From fuels ▪ Animals ▪ Most bacteria The molecular logic of life We look at the chemistry that is behind the: ▪ Initiation and acceleration of reactions ▪ Organization and specificity of metabolism and signaling ▪ Storage and transfer of information and energy There is a hierarchy of structure (simple-to- complex) 30 elements essential for life Other than carbon, elements H, O, N, P, and S are also common. Metal ions (e.g., K+, Na+, Ca++, Mg++, Zn++, Fe++) play important roles in metabolism. Biochemistry: Unique role of carbon Biochemistry is simply a specialized area of organic chemistry Carbon is of the utmost importance and extremely versatile, due to its ability to form single, double, and triple bonds with itself and other elements/groups Common functional groups of biological molecules There are a limited number of “functional groups” found in biomolecules Each group has unique properties, which are retained independently, yet also Influence the physical and chemical properties of the molecule to which they are attached Biological molecules typically have several functional groups Complex (and even relatively simple ones) often have multiple functional groups, each rendering a specific function The ABCs of life Some representative organic compounds that are the basis for more complex macromolecules Function of molecules depends on 3-D structure (isomers) Stereoisomers ▪ Can have different physical properties ▪ Cannot be interconverted without breaking bonds Geometric isomers (cis vs. trans) ▪ See example on right (arrangement around double bond) ▪ Have different physical and chemical properties Enantiomers (mirror images) ▪ Have identical physical properties (except with regard to polarized light) and react identically with achiral reagents Diastereomers ▪ Have different physical and chemical properties An important biochemical example of cis vs. trans Retinal is found in the vertebrate retina ▪ Normally in the 11-cis- retinal form Upon absorption of light (energy), converted to all- trans-retinal ▪ This triggers an electrical change that is registered as a nerve impulse Enantiomers and diastereomers Isomers have same chemical formula, but arrangement of atoms differs Enantiomers are mirror images Diastereomers are non- mirror images Enantiomers A two amino acid peptide differs in its effect (in this case, taste) in humans The arrangement around a single chiral carbon determines whether the dipeptide is sweet or bitter Interactions between biomolecules are specific Macromolecules fold into 3D structures with unique binding pockets. Only certain molecules fit in well and can bind. In living organisms, chiral molecules are usually only present on ONE of their forms ▪ Example: amino acids are only present as the L-isomer ▪ Example: glucose occurs only as its D- isomer Binding of chiral biomolecules is stereospecific. Genetic and evolutionary foundations Life on Earth arose 3.5–3.8 billion years ago. The formation of self-replicating molecules was a key step. Could it have been DNA? Could it have been proteins? RNA world? RNA can act both as the information carrier and biocatalyst. Some viruses use RNA as a primary means of genetic information. One proposed “RNA world” scenario Complementarity in DNA allows for near-perfect replication Complementarity in DNA allows for replication with near-perfect fidelity Near-perfect, but not perfect, allows for changes in sequence that can allow for natural selection to occur Central dogma of biochemistry: DNA → RNA → protein For the most part, this is a one-way pathway ▪ This is especially true once a mRNA is translated into a protein Natural selection favors some mutations How can natural selection act on “near- perfect” DNA replication? Mutations occur more or less randomly in DNA and RNA. Mutated polynucleotides may be transcribed and translated into molecular machinery like proteins. Mutations that give organisms an advantage in a given environment are more likely to be propagated. An example of gene duplication and mutation to encode for a “new” protein is shown on right Evolution of eukaryotes likely also mediated through endosymbiosis Mitochondria and chloroplasts were likely once prokaryotic organisms that have been subsequently co-opted into eukaryotic cells to perform specialized functions in those cells. Chapter 13 Introduction to Metabolism © 2021 Macmillan Learning Laws of thermodynamics apply to living organisms Energy cannot be created or destroyed (conservation of energy). The disorder of a system always increases (entropy) ▪ Living organisms cannot create energy from nothing. ▪ Living organisms cannot destroy energy into nothing. ▪ Living organisms may transform energy from one form to another. In the process of transforming energy, living organisms must increase the entropy of the universe. In order to maintain organization within themselves, living systems must be able to extract useable energy from their surroundings and release “useless” energy (heat) back to their surroundings Organisms perform energy transductions to accomplish work Living organisms exist in a dynamic steady state and are never at equilibrium with their surroundings. Energy coupling allows living organisms to transform matter into energy. Biological catalysts reduce energy requirement for reactions while offering specificity. As the entropy of the universe increases, creating and maintaining order requires work and energy The laws of thermodynamics apply to living organisms So if energy cannot be created (or destroyed), where do living organisms get energy from? ▪ Three types of systems in thermodynamics: - Open system – exchange both energy and matter with surroundings - Closed system – exchange only energy with surroundings - Isolated system – cannot exchange energy or matter with surroundings Organisms perform energy transductions to accomplish work Energy can neither be created nor destroyed... ▪ But it can be transformed/transduced to perform “work” Life needs energy Recall that living organisms are built of complex structures. Building complex structures that are low in entropy is only possible when energy is spent in the process. The ultimate source of this energy on Earth is sunlight Metabolism is the sum of all chemical reactions in the cell Some chemical reactions in cells release energy, while others require energy These are intertwined, as release of energy from one reaction can drive a reaction that requires that energy When we consider the sum of all reactions in a cell, this is the metabolism of a cell (two basic forms): ▪ Anabolism - making complex molecules from simpler ones (requires energy) ▪ Catabolism - breaking down complex molecules into simpler ones (releases energy) Gibbs free-energy The free-energy (G) content of any closed Entropy (S) is the randomness or disorder system is defined by the quantities of: of a system ▪ Entropy (S), ▪ More disorder = positive S value ▪ Enthalpy (H), and ▪ Less disorder = negative S value ▪ Absolute temperature (T; in Kelvin) Free energy is defined by the equation: Enthalpy (H) is the sum of a system’s G = H – TS internal energy The free-energy change (G) when a ▪ This is reflected by the numbers and kinds chemical reaction occurs at a constant of bonds that are broken and formed temperature is determined by the change ▪ Positive H value = endothermic (absorb heat energy from surroundings) in enthalpy (H) and entropy (S), such ▪ Negative H value = exothermic (release that: thermal energy to surroundings) G = H – TS Equilibrium and G measure the spontaneity of a reaction For the equation: G is the standard free-energy change; a aA + bB ⇌ cC + dD constant characteristic for a specific reaction at standard conditions ▪ Temp = 25C Equilibrium constant calculated as: ▪ Pressure = 1.0 atm [C]ceq [D]d eq ▪ [Reactants] = 1 M Keq = [A]aeq [B]b eq G (or G) are often used to Free energy calculated as:  distinguish biochemistry from physical chemistry free-energy changes G = −RTlnKeq ▪ Same as for G, except that... ▪ pH = 7.0 (near physiological conditions) ▪ R = 8.314 J/molK ▪ T = absolute temperature in Kelvin (K) Derivation of G Gibbs showed that G is a function of the standard free-energy change Go G = G + RTlnKeq G (the actual free energy change) for any chemical reaction is a function of BOTH the standard free-energy change (a constant specific to each chemical reaction) AND the initial concentrations of reactants and products ▪ Quite simply: G is really a measurement of the distance of a system from its equilibrium position (where no driving force remains/no work can be done) Substituting zero for G (in the above equation), we get the measure of (equation for) standard free-energy (as shown on the previous slide) ▪ G is an alternative way (besides Keq) to express the driving force of a reaction...in terms of energy Free energy, or the equilibrium constant, determines spontaneity When G is negative, this is an exergonic reaction ▪ The reaction can occur spontaneously When G is positive, this is an endergonic reaction ▪ The reaction does NOT occur spontaneously Also, note how Keq and G are related Free energy, or the equilibrium constant, determines spontaneity NOTE: Thermodynamic constants such as these show where the final equilibrium lies and indicates spontaneity of reactions; however... ▪ This has NO bearing on how fast (the rate) that equilibrium will actually be achieved (covered in Ch 6: Kinetics) Unfavorable and favorable reactions Synthesis of complex molecules and many other metabolic reactions requires energy (endergonic). ▪ A reaction might be thermodynamically unfavorable (G > 0) - Creating order requires work and energy ▪ A metabolic reaction might have too high an energy barrier (G‡ > 0) - Metabolite is kinetically stable The breakdown of some metabolites releases a significant amount of energy (exergonic) ▪ Such metabolites (ATP, NADH, NADPH) can be synthesized using the energy from sunlight and fuels ▪ Their cellular concentration is far higher than their equilibrium concentration Standard free-energy changes and energy coupling Energy coupling Chemical coupling of exergonic and endergonic reactions allows otherwise unfavorable reactions The “high-energy” molecule (ATP) reacts directly with the metabolite that needs “activation” Energetics within the cell The actual free-energy change of a reaction in the cell depends on the: ▪ Standard biochemical change in free energy (G) ▪ Actual concentrations of products and reactants For the reaction: aA + bB ⇌ cC + dD CcD d G = G + RTlnKeq = G + RTln a b A B Standard free-energy changes are additive: Reaction 1: A⇀C G1 Reaction 2: B⇀D G2 Sum: A+B⇀C+D G1 + G2 How to speed up reactions Higher temperatures Change the reaction by ▪ Stability of macromolecules is coupling to a fast one limiting Lower activation barrier by Higher concentration of catalysis reactants ▪ Both universally used by living ▪ Costly, as more valuable organisms starting material is needed Enzymes (catalysts):  activation energy and  reaction rate Many chemical reactions, including those that breakdown macromolecules into monomers, occur very slowly A catalyst is a compound that increases the rate of a chemical reaction Catalysts do not alter G Catalysts lower the activation free energy G‡ Enzymatic catalysis offers: ▪ Acceleration under mild conditions (sometimes 1012 times faster) ▪ High specificity ▪ Possibility for regulation Biochemical pathway: Series of related enzyme-catalyzed rxns Metabolic pathway ▪ produces energy or valuable materials ▪ anabolic (synthesis) or catabolic (breakdown) Signal transduction pathway ▪ transmits information Reminder: Metabolism Metabolism is the sum of all chemical reactions in the cell/organism ▪ But what if there is ample molecule around…no need to synthesize? ▪ But what if there is a shortage of reactants…no need to breakdown/convert? Must be a way for cells to prevent unnecessary use of energy when pathway is not needed, right? Pathways are controlled in order to regulate levels of metabolites Example of a negative regulation (feedback inhibition): Product of enzyme 5 inhibits enzyme 1 to prevent wasteful excess products. There are thousands of intermediates and numerous interdependent pathways in the cell Chapter 13: Summary The rules of thermodynamics and organic chemistry still apply to living systems Reactions are favorable when the free energy of products is much lower than the free energy of reactants Unfavorable reactions can be made possible by chemically coupling a highly favorable reaction to the unfavorable reaction Be sure to think about how H, S, and Keq relate to G (or G°) Chapter 2 Water, the Solvent of Life © 2021 Macmillan Learning Chapter: Water, the solvent of life Learning goals: ▪ What kind of interactions occur between molecules ▪ Why water is a good medium for life ▪ Why nonpolar moieties aggregate in water ▪ How dissolved molecules alter properties of water ▪ How weak acids and bases behave in water ▪ How buffers work and why we need them ▪ How water participates in biochemical reactions Water is the medium for life Life evolved in water due to the protection it provides from UV light and has a high heat of vaporization. Organisms typically contain 70-90% water. Chemical reactions occur in aqueous milieu. Water is a critical determinant of the structure and function of proteins, nucleic acids, and membranes Structure of the water molecule The octet rule dictates that there are four electron pairs around an oxygen atom in water. These electrons are in four sp3 orbitals. Two of these pairs covalently link two hydrogen atoms to a central oxygen atom. The two remaining pairs remain nonbonding (lone pairs). Water geometry is a distorted tetrahedron. The electronegativity of the oxygen atom induces a net dipole moment. Because of the dipole moment, water can serve as both a hydrogen bond donor and acceptor Hydrogen bonds Hydrogen bonds are strong dipole- dipole or charge-dipole interactions that arise between a covalently bound hydrogen and lone pair of electrons. ▪ They typically involve two electronegative atoms (frequently nitrogen and oxygen). Hydrogen bonds are strongest when the bonded molecules allow for linear bonding patterns. ▪ Ideally, the three atoms involved are in a line Hydrogen bonding in water The hydrogen (H) bond (as it applies to water) = electrostatic attraction between the oxygen atom of one water molecule and the hydrogen of another Water can serve as both: ▪ An H donor ▪ An H acceptor Up to four H-bonds per water molecule gives water its: ▪ Anomalously high boiling point ▪ Anomalously high melting point ▪ Unusually large surface tension Hydrogen bonding in water is cooperative. Hydrogen bonds between neighboring molecules are weak (20 kJ/mol) relative to the H-O covalent bonds (420 kJ/mol) Ice: Water in a solid state Water has many different crystal forms; the hexagonal ice is the most common. Hexagonal ice forms an organized lattice and thus has a low entropy. Hexagonal ice contains maximal hydrogen bonds/ water molecules, forcing the water molecules into equidistant arrangement. Thus: ▪ Ice has lower density than liquid water ▪ Ice floats In ice, each H2O molecule forms 4 hydrogen bonds with other H2O molecules Importance of hydrogen bonds Source of unique properties of “I believe that as the methods of structural water chemistry are further applied to physiological problems, it will be found that Structure and function of proteins the significance of the hydrogen bond for Structure and function of DNA physiology is greater than that of any other Structure and function of single structural feature.” polysaccharides –Linus Pauling, The Nature of the Chemical Bond, 1939 Binding of substrates to enzymes Binding of hormones to receptors Matching of mRNA and tRNA Biological relevance of hydrogen bonds Water as a solvent Water is a good solvent for charged and Water is a poor solvent for nonpolar polar substances: substances: ▪ Amino acids and peptides ▪ Nonpolar gases ▪ Small alcohols ▪ Aromatic moieties ▪ Carbohydrates ▪ Aliphatic chains Water forms hydrogen bonds with polar solutes Hydrogen bonds readily form between an electronegative atom (the hydrogen acceptor) and a hydrogen atom covalently bonded to another electronegative atom (the hydrogen donor) Nonpolar gases are poorly soluble in water Biologically important gases CO2, O2, N2 are nonpolar Their movement into aqueous solution decreases entropy by constraining their motion Physics of noncovalent Noncovalent interactions do not involve sharing a pair of electrons. Based on their physical origin, interactions one can distinguish between: ▪ Ionic (Coulombic) interactions - Electrostatic interactions between permanently charged species, or between the ion and a permanent dipole ▪ Dipole interactions - Electrostatic interactions between uncharged but polar molecules ▪ van der Waals/London dispersion interactions - Weak interactions between all atoms, regardless of polarity - Attractive (dispersion) and repulsive (steric) component ▪ Hydrophobic Effect - Complex phenomenon associated with the ordering of water molecules around nonpolar substances Dissolving salts involves breaking ionic interactions Crystal lattice of salts disrupted into ions that make up the salt H2O molecules cluster about the ions, keeping separated and in solution (dissolved) ▪ Ions are said to be hydrated Nonpolar compounds in water force unfavorable energetics Nonpolar compounds interfere with the H2O molecules form a highly ordered, hydrogen bonding among H2O molecules cage-like shell around each nonpolar ▪ Increases enthalpy (H) and decreases solute molecule entropy (S) ▪ maximizes solvent-solvent hydrogen bonding The free-energy change (G = H − TS) ▪ H2O molecules are not as highly arranged as those in clathrates (crystalline compounds for dissolving a nonpolar solute in water is of nonpolar solutes and water) unfavorable: ▪ H has a positive value ▪ S has a negative value ▪ G has a positive value Origin of the hydrophobic effect Consider amphipathic lipids in water. Lipid molecules disperse in the solution; nonpolar tails of lipid molecules are surrounded by ordered water molecules. Entropy of the system decreases. The system is now in an unfavorable state. Origin of the hydrophobic effect Nonpolar portions of the amphipathic molecule aggregate, such that fewer water molecules are ordered and entropy increases. All nonpolar groups are sequestered from water, and the released water molecules increase the entropy further. Only polar “head groups” are exposed Origin of the hydrophobic effect With high enough concentration of amphipathic molecules, complete aggregation into micelles is possible Hydrophobic effect favors ligand binding Binding sites in enzymes and receptors are often hydrophobic. Such sites can bind hydrophobic substrates and ligands, such as steroid hormones, which displace water and increase entropy of the system. Many drugs are designed to take advantage of the hydrophobic effect. van der Waals interactions van der Waals interactions have Biochemical significance of vdW two components: interactions: ▪ Attractive force (London ▪ Weak individually dispersion), which depends on - Easily broken, the polarizability reversible ▪ Repulsive force (Steric ▪ Universal repulsion), which depends on - Occur between any the size of atoms two atoms that are Attraction dominates at longer near each other distances (typically 0.4-0.7 nm). ▪ Importance Repulsion dominates at very short - Determines steric distances. complementarity There is a minimum energy - Stabilizes biological distance (van der Waals contact macromolecules distance) (stacking in DNA) - Facilitates binding of polarizable ligands Weak interactions are crucial to macromolecular structure Noncovalent interactions are much weaker than covalent bonds Continually forming and breaking Dictate macromolecular structure, which ultimately affects function ▪ For macromolecules, the most stable structure usually maximizes weak interactions Solutes can change the properties of water Colligative properties: ▪ Boiling point, melting point, and osmolarity ▪ Do not depend on the nature of the solute, just the concentration Noncolligative properties: ▪ Viscosity, surface tension, taste, and color ▪ Depend on the chemical nature of the solute Cytoplasm of cells are highly concentrated solutions and have high osmotic pressure due to dissolved solutes Concentrated solutes produce osmotic pressure Solutes alter the colligative properties of Movement of water from higher to lower the solvent: concentrations produces osmotic ▪ Vapor pressure pressure ▪ Boiling point ▪ Melting point (freezing point) ▪ Osmotic pressure Effect depends on the number of solute particles (molecules or ions) in a given amount of water Calculating osmotic pressure Osmotic pressure () is: Osmotic pressure () depends on: ▪ The force necessary to resist water movement ▪ The van’t Hoff factor, a measure of the ▪ Approximated by the van’t Hoff equation extent to which the solution dissociates into 2+ ionic species (i)  = 𝑖𝑐𝑅𝑇 o For non-ionizing solutes, i = 1 o For solutes that dissociate into two ions, i = 2 ▪ The solute’s molar concentration (c) ▪ R is the gas constant and T is the absolute temperature Osmolarity, osmosis, and effects on cells Osmolarity (ic) = the product of the van’t Hoff factor (i) and the solute’s molar concentration (c) Osmosis = water movement across a semipermeable membrane driven by differences in osmotic pressure Concentration of solutes outside of or inside of cell can have pressure effects on cell itself Pure water is slightly ionized H-O covalent bonds are polar and can Hydrogen ions are immediately hydrated dissociate heterolytically to form hydronium ions (H3O+): The products are a proton (H+) and a Most water molecules remaining un- ionized, thus pure water has very low hydroxide ion (OH-) electrical conductivity (resistance = 18 M cm) H2O molecules have a slight tendency to undergo reversible ionization to yield a Also, the equilibrium is strongly to the left hydrogen ion (a proton) and a hydroxide (low Keq) ion: H2O ⇌ H+ + OH− (Eqn 2-1) Proton hydration and hopping Protons do not exist free in solution “Proton hopping” results in high ionic mobility ▪ They are immediately hydrated to form hydronium ions (H3O+) A hydronium ion is a water molecule with a proton associated with one of the nonbonding electron pairs Hydronium ions are solvated by nearby water molecules. The covalent and hydrogen bonds are interchangeable, allowing for an extremely fast mobility of protons in water via “proton hopping” Water can be essential to protein function Example: Cytochrome f: ▪ Has a chain of five bound H2O molecules ▪ This may provide a path for protons to move through the membrane Ionization of water: Quantitative treatment H2O ⇌ H+ + OH− Concentrations of participating species in an equilibrium process are not independent but are related via the equilibrium constant: H+ [OH−] Keq = [H2O] ▪ Keq can be determined experimentally...it is 1.8x10–16 M @ 25C ▪ [H2O] can be determined from the density of water...it is 55.5 M Ionic product of water: Kw = Keq H2O = H+ OH− = 55.5 M 1.8×10−16 M = 1×10−14 M2 In pure water (neutral), [H+] = [OH-] = 10-7 M The pH scale designates the H+ and OH- concentrations “p” is defined as the negative logarithm of something - used to simplify numbers The pH scale is based on the ion product of water, Kw Kw = [H+][OH−] = 1x10−14 M2 − log H+ − log OH− = +14 pH + pOH = 14 For a precisely neutral solution at 25°C, pH = 7.0 (i.e., [H+] = [OH-] = 10-7) The pH and pOH must always add up to 14. pH can be negative ([H+] = 6 M) pH scale is logarithmic: 1 unit = 10-fold Just want to emphasize this point. The pH of some aqueous fluids pH values > 7: ▪ Alkaline (also referred to as basic) ▪ Concentration of OH- is greater than that of H+ pH values < 7: ▪ Acidic ▪ Concentration of H+ is greater than that of OH- pH and medical diagnoses Acidosis = pH of blood plasma below the normal value of 7.4 ▪ Common in people with severe, uncontrolled diabetes ▪ Caused by accumulation of high concentrations of two carboxylic acids, - hydroxybutyric acid and acetoacetic acid - Dissociation of these acids lowers the pH of blood plasma to less than 7.35 Alkalosis = pH of blood plasma above the normal value of 7.4 Extreme acidosis or alkalosis can be life-threatening ▪ Severe changes in pH causes enzymes to not function properly Dissociation of weak electrolytes: principle O Weak electrolytes dissociate only + H2O Keq O H3C H3C + H3O+ partially in water. O- OH The extent of dissociation is determined by the acid dissociation constant Ka. [H + ][CH3 COO− ] We can calculate the pH if the Ka is 𝐾𝑎 = = 1.74 ⋅ 10−5 M [CH3 COOH] known. But some algebra is needed! Dissociation of weak electrolytes: example What is the final pH of a solution O O Ka H3C H3C + H+ when 0.1 moles of acetic acid is added to water to a final volume of OH O- 1L? 0.1 – x x x ▪ We assume that the only source of H+ is the weak acid. [x][x] 𝐾𝑎 = = 1.74 ⋅ 10−5 M ▪ To find the [H+], a quadratic [0.1−x] equation must be solved. x 2 = 1.74 ⋅ 10−6 − 1.74 ⋅ 10−5 x x 2 + 1.74 ⋅ 10−5 x − 1.74 ⋅ 10−6 = 0 x = [H+] = 0.001310; therefore, pH = 2.883 Dissociation of weak electrolytes: simplification The equation can be simplified if O O Ka the amount of dissociated species is H3C H3C + H+ OH O - much less than the amount of undissociated acid. 0.1 – x x x 0.1 x x Approximation works for sufficiently [x][x] weak acids and bases. 𝐾𝑎 = = 1.74 ⋅ 10−5 M [0.1] Check that x < [total acid] x 2 = 1.74 ⋅ 10−6 x = [H+] = 0.00132; therefore, pH = 2.880 Weak acids/bases: characteristic acid dissociation constants Conjugate acid-base pair Common conjugate acid-base pairs: = a proton donor and its corresponding proton acceptor The stronger the acid, the greater its tendency to lose its proton pKa measures acidity: pKa = -log Ka Actual pKa values are determined experimentally Titration curves reveal the pKa of weak acids (like acetic acid) Titration curve = a plot of pH against the amount of NaOH added At the midpoint, the pH of the equimolar solution = the pKa of the weak acid (acetic acid shown to right) Buffers are mixtures of weak acids and their anions (conjugate base) Buffers resist change in pH. At pH = pKa, there is a 50:50 mixture of acid and anion forms of the compound. Buffering capacity of acid/anion system is greatest at pH = pKa. Buffering capacity is lost when the pH differs from pKa by more than 1 pH unit Weak acids have different pKa’s, and hence, differ in buffering The Henderson-Hasselbalch Consider the following ionization of a weak acid: equation Relates pH, pKa, and buffer concentration HA ⇌ H+ + A− Derivation of the Henderson-Hasselbalch Henderson-Hasselbalch equation = equation: describes the shape of the titration curve [H+][A−] Ka = of any weak acid [HA] [HA] [H+] = Ka [A−] [A−] pH = pKa + log [HA] (Eqn 2-9) [HA] −log[H+] = −logKa– log − [A ] [HA] pH = pKa − log [A−] [A−] pH = pKa + log (Eqn 2-9) [HA] Henderson-Hasselbalch equation: Example A buffer is comprised of 0.1 M acetic acid (CH3COOH; pKa = 4.76) and 0.05 M sodium acetate (CH3COO-Na+). What is the final pH of the buffer? You know the following: pKa = 4.76 [HA] = 0.1 M [A-] = 0.05 M Plug into the H-H equation: pH = pKa + log [A-]/[HA] = 4.76 + log (0.05 M/0.1 M) = 4.45 Biological Buffer Systems Maintenance of intracellular pH is vital to all cells. ▪ Enzyme-catalyzed reactions have optimal pH. ▪ Solubility of polar molecules depends on H-bond donors and acceptors. ▪ Equilibrium between CO2 gas and dissolved HCO3– depends on pH. HO Buffer systems in vivo are mainly based on: N N ▪ Phosphate, concentration in millimolar range ▪ Bicarbonate, important for blood plasma S O3Na ▪ Histidine, efficient buffer at neutral pH Buffer systems in vitro are often based on sulfonic acids of cyclic amines. ▪ HEPES ▪ PIPES ▪ CHES The bicarbonate buffer system involves 3 reversible equilibria The pH of a bicarbonate buffer system exposed to a gas phase depends on: ▪ The concentration of HCO3- ▪ The partial pressure of CO2 (pCO2) = the concentration of CO2 in the gas phase Buffer system is effective near pH 7.4 Calculating blood pH The rate of respiration (controlled by the ▪ Typical values: brain stem) can quickly adjust these - [HA] = [H2CO3] = 1.2 mM equilibria to keep the blood pH nearly - [A-] = plasma [HCO3-] = 24 mM constant - pKa = 6.1 Hyperventilation lowers aqueous CO2, ▪ Therefore, blood pH… which raises blood pH = pKa + log [A-]/[HA] = 6.1 + log (24 mM/1.2 mM) = 7.4 Biologically-relevant buffers: The phosphate buffer system The phosphate buffer system acts in the cytoplasm of all cells: H2PO4− ⇌ H+ + HPO42− H2PO4− acts as a proton donor and HPO42− acts as a proton acceptor Buffer system is maximally effective at a pH close to its pKa of 6.86 ▪ Works over a range between 5.9 and 7.9 An example of buffering capacity H What if there were no buffering capacity? Chapter 2: Summary In this chapter, we learned about the: ▪ Nature of intermolecular forces ▪ Properties and structure of liquid water ▪ Behavior of weak acids and bases in water ▪ Way water can participate in biochemical reactions ▪ Dissociation of weak acids ▪ Buffers and how they achieve their buffering capacity Chapter 3 Amino Acids, Peptides, and Proteins © 2021 Macmillan Learning Chapter 3: Amino acids, peptides, and proteins Learning goals: ▪ Structure and naming of amino acids ▪ Structure and properties of peptides ▪ Ionization behavior of amino acids and peptides ▪ Methods to characterize peptides and proteins Proteins: The primary agents of a wide range of biological functions Catalysis ▪ Enolase (in the glycolytic pathway) ▪ DNA polymerase (in DNA replication) Transport ▪ Hemoglobin (transports O2 in the blood) ▪ Lactose permease (transports lactose across the cell membrane) Structure ▪ Collagen (connective tissue) ▪ Keratin (hair, nails, feathers, horns) Motion ▪ Myosin (muscle tissue) ▪ Actin (muscle tissue, cell motility) Amino acids: The building blocks of proteins Proteins are linear heteropolymers of -amino acids. Amino acids have properties that are well suited to carry out a variety of biological functions: ▪ Capacity to polymerize ▪ Useful acid-base properties ▪ Varied physical properties ▪ Varied chemical functionality Amino acids share many features, differing only at the R substituent 20 common amino acids (aa) All are  (alpha)-amino acids Amino group and carboxyl group both bonded to the same carbon (-carbon) Differ from each other in their side chains (R groups), which vary in: ▪ Size ▪ Structure ▪ Electric charge ▪ Solubility (in water) ▪ This general structure is common for every amino acid, except proline (cyclic) Amino acids: Three common functional groups The α carbon always has four substituents and is tetrahedral. All (except proline) have: ▪ An acidic carboxyl group connected to the α carbon ▪ A basic amino group connected to the α carbon ▪ An  hydrogen connected to the α carbon The fourth substituent (R) is unique in glycine, the simplest amino acid, where it is also a hydrogen All amino acids are chiral (except glycine) Amino acids have two possible stereoisomers (or more specifically, enantiomers; non-superposable mirror images) Molecules (in this case, amino acids) are optically active; however… Nomenclature based on configuration compared to L-/D-glyceraldehyde Proteins only contain l-amino acids Key conventions in designating amino acids by name (examples) Three-letter code is fairly simple 3 Letter 1 Letter (usually, but not always, the first Name Code Code three letters of aa) Alanine Ala A One should be able to identify an Aspartate Asp D amino acid in a sequence by either Asparagine Asn N the one- or three-letter code. Tryptophan Trp W Glycine Gly G Glutamate Glu E Glutamine Gln Q 111 Amino acids: Classification There are a number of different 1. Nonpolar (hydrophobic), classification schemes that can be aliphatic (7 aa) used. 2. Aromatic (3 aa) For this class (and the textbook), amino acids can be classified into 3. Polar (hydrophilic), uncharged five main classes (based on (5 aa) properties of their R-groups). 4. Positively-charged (3 aa) 5. Negatively-charged (2 aa) Nonpolar (hydrophobic), aliphatic R-groups Side chains of A, V, L, and I tend to cluster together within proteins (hydrophobic effect) Glycine has simplest structure, no real contribution to hydrophobic effect Methionine has sulfur (slight nonpolar thioether group) Proline has distinctive cyclic structure, with am imino group reducing its structural flexibility Aromatic R-groups All contribute to the hydrophobic effect OH-group in Tyrosine can form hydrogen bonds Tyrosine and Tryptophan are more polar due to hydroxyl and nitrogen (in the indole ring) NOTE: These amino acid side chains absorb UV light at 270–280 nm (although Phe to a much lesser extent) Polar (hydrophilic), uncharged R- groups These amino acids side chains can form hydrogen bonds Cysteine can form disulfide bonds through oxidation between sulfhydryl groups ▪ Disulfide-linked residues are strongly hydrophobic Asparagine and Glutamine are the amides of Aspartate and Glutamate Negatively-charged R-groups Second carboxyl group (COO-) contributes to negative charge at pH 7.0 Positively-charged R-groups Lysine: second primary amino group Arginine: positively-charged guanidinium group Histidine: aromatic imidazole group (can be protonated at pH 7.0) ▪ His residues facilitate many enzyme- catalyzed reactions by serving as proton donors/acceptors Uncommon amino acids also have important functions Modifications of common amino acids: ▪ Modified after protein synthesis e.g., 4-hydroxyproline, found in collagen ▪ Modified during protein synthesis e.g., pyrrolysine, contributes to methane biosynthesis ▪ Modified transiently to change protein’s function e.g., phosphorylation Free metabolites ▪ e.g., ornithine, intermediate in arginine biosynthesis Some properties of amino acids Amino acids vary in molecular weight (Mr), except for leucine and isoleucine They also differ in hydropathy index (the measure of the interaction with water) ▪ The more negative the hydropathy index, the more that aa interacts with H2O ▪ Notice that these aa’s are either charged or contain a polar group Amino acids vary in their overall occurrence in proteins All amino acids are ionizable, and some even have ionizable side chains (R-groups) 119 Ionization of amino acids All amino acids contain at least two ionizable protons, each with its own pKa. The carboxylic acid group has an acidic pKa: −COOH ⇌ −COO− + H+ The amino group has a basic pKa: −NH4+ ⇌ NH3 + H+ Amino acids can act as acids or bases At low pH, the amino acid exists in a positively charged form (cation). At high pH, the amino acid exists in a negatively charged form (anion). Between the pKa for each group, the amino acid exists in a zwitterion form, in which a single molecule has both a positive and negative charge. Chemical environment affects pKa values -carboxy group is much more acidic than in carboxylic acids. -amino group is slightly less basic than in amines. Information gleaned from titration of amino acids Cation → Zwitterion → Anion Amino acids act as buffers: ▪ Reminder: Buffers prevent changes in pH close to the pKa ▪ Glycine has two buffer regions: Centered around the pKa of the - carboxyl group (pK1 = 2.34) Centered around the pKa of the -amino group (pK2 = 9.6) Cation ⇌ Zwitterion ⇌ Anion ▪ -COOH has an acidic pKa (pK1) ▪ -NH3+ has a basic pKa (pK2) The pH at which the net electric charge is zero is the isoelectric point (pI) Isoelectric point, pI Cation → Zwitterion → Anion Zwitterions predominate at pH values between the pKa values of the amino- and carboxyl-groups. For amino acids without ionizable side chains (like glycine on the previous slide), the isoelectric point (pI) is: pK1 + pK2 pI = 2 ▪ pH = pI = net charge is zero (amino acid least soluble in water, does not migrate in electric field) ▪ pH > pI = net negative change ▪ pH < pI = net positive charge Titration of amino acids with an ionizable R group What if amino acids have an ionizable side chain? Ionizable side chains: ▪ Have a pKa value ▪ Act as buffers ▪ Influence the pI of the amino acid ▪ Can also be titrated (titration curve has 3 ionization steps) Titration curves are now more complex, as each pKa has a buffering zone of 2 pH units, and each aa has multiple buffering zones. How to calculate the pI when the side chain is ionizable Identify species that carries a net zero charge. Identify the pKa value that defines the acid strength of this zwitterion: (pKR). Identify the pKa value that defines the base strength of this zwitterion: (pK2). Take the average of these two pKa values. What is the pI of histidine? Calculating the pI of histidine Identify the pKa value that defines the acid strength of this zwitterion: (pKR). Identify the pKa value that defines the base strength of this zwitterion: (pK2). Take the average of these two pKa values. What is the pI of histidine? Practice: Calculating the pI of glutamate What is the pI of glutamate? Amino acids polymerize to form peptides Peptides are small condensation products of amino acids. They are “small” compared with proteins (MW < 10 kDa) Peptide bond: ▪ Covalent ▪ Formed through condensation ▪ Broken through hydrolysis Peptide ends are not the same Numbering (and naming) starts from the amino AA1 AA2 AA3 AA4 AA5 terminus (N-terminal). Peptide conventions and nomenclature Dipeptide = 2 amino acids, 1 peptide bond Numbering (and naming) starts from the amino- terminal residue (N-terminal) Tripeptide = 3 amino acids, 2 peptide bonds Oligopeptide = a few amino acids Polypeptide = many amino acids, molecular weight < 10 kDa N-terminus C-terminus Protein = thousands of amino acids, molecular weight > 10 kDa Full amino acid names: ▪ serylglycyltyrosylalanylleucine Three-letter code abbreviations: ▪ Ser-Gly-Tyr-Ala-Leu One-letter code abbreviation: ▪ SGYAL Peptides can be distinguished by their ionization behavior Ionizable groups in peptides: ▪ One free -amino group (N- terminus) ▪ One free -carboxyl group (C- terminus) ▪ Some R groups (internal and variable) Peptides have biological roles and distinct properties Artificial sweetener (aspartame) Peptides: A variety of functions Hormones and pheromones 11 ▪ Insulin (think sugar metabolism) ▪ Oxytocin (think childbirth) 10 12 ▪ Sex-peptide (think fruit fly mating) 9 Neuropeptides ▪ Substance P (pain mediator) Antibiotics 2 4 6 8 1 ▪ Polymyxin B (for Gram – bacteria) ▪ Bacitracin (for Gram + bacteria; 7 shown on right) 3 5 Protection, e.g., toxins Bacitracin ▪ Amanitin (mushrooms) 1 2 3 4 5 6 7 8 9 10 11 12 L-Ile|L-Cys|L-Leu|D-Glu|L-Ile|L-Lys|D-Orn|L-Ile|D-Phe|L-His|D-Asp|L-Asn ▪ Conotoxin (cone snails) ▪ Chlorotoxin (scorpions) Conjugated proteins: Covalently bound to a nonprotein entity Polypeptides (covalently linked -amino acids) + possibly: ▪ Cofactors - Functional non-amino acid component - Metal ions or organic molecules ▪ Coenzymes - Organic cofactors - NAD+ in lactate dehydrogenase ▪ Prosthetic groups ▪ Covalently attached cofactors ▪ Heme in myoglobin ▪ Other modifications (posttranslational modifications) Uncommon amino acids: Most often generated by PTM Uncommon amino acids often found specific tissues and play special roles (e.g., clotting factors) Selenocysteine and pyrrolysine are ones that are introduced during protein synthesis (by ribosome during translation) Many modifications can occur transiently to alter protein function (e.g., phosphorylation) and are important in regulation and signaling Peptide subunits and amino acid composition of proteins Multi-subunit protein = 2+ polypeptides associated noncovalently Oligomeric protein = at least 2 identical subunits ▪ identical units = protomers Amino acid composition is highly variable from protein-to-protein Biologically-active (poly)peptides: sizes and compositions Length of naturally occurring peptides = 2 to many thousands of amino acid residues Estimating the number of amino acid residues: ▪ Number of residues = molecular weight (often in kDa, so need to convert to Da)/110 Da ▪ Average molecular weight of amino acid = ~128 Da ▪ Molecule of water removed to form peptide bond = 18 Da ▪ Thus, 128 - 18 = 110 Da/residue Common questions about peptides and proteins What is its sequence and composition? What is its three-dimensional structure? How does it find its native fold? How does it achieve its biochemical role? How is its function regulated? How does it interact with other macromolecules? How is it related to other proteins? Where is it localized within the cell? What are its physico-chemical properties? Studying proteins: Separation and purification from a mixture Polypeptides contain differing amino acid Separation relies on differences in sequences. physical and chemical properties: The sequence and arrangement of amino ▪ Charge acids gives the polypeptide a chemical ▪ Size character (i.e., charged, polar, ▪ Affinity for a ligand hydrophobic, etc.). ▪ Solubility Some polypeptides bind specific targets, ▪ Hydrophobicity which can be used to “fish them out” of a ▪ Thermal stability complex mixture. Chromatography is commonly used for preparative separation in which the protein is often able to remain fully folded Column chromatography Column chromatography allows separation of a mixture of proteins over a solid phase (porous matrix) using a liquid phase to mobilize the proteins. Proteins with a lower affinity for the solid phase will wash off first; proteins with higher affinity will retain on the column longer and wash off later. Separation by charge: Ion exchange chromatography Separates based on sign and magnitude of the net electric charge pH and concentration of free salt ions affect protein affinity Uses bound charged groups: ▪ Cation exchangers ▪ Anion exchangers Separation by size: Size exclusion (gel filtration) chromatography Also called gel filtration chromatography Separates based on size Large proteins emerge from the column before small proteins do Separation by binding: Affinity Separates based on binding affinity Eluted by high concentration of salt or ligand High-performance liquid chromatography (HPLC) Uses high-pressure pumps to move proteins down the column Greatly improves resolution https://www.creative-proteomics.com/pronalyse/high-performance-liquid-chromatography-service.html Sequential purification steps decrease sample size Purification factor = final specific activity/starting specific activity Percent yield = percentage of the final activity/percentage starting activity Electrophoresis for protein analysis Separation in analytical scale is commonly done by electrophoresis. ▪ The electric field pulls proteins according to their charge. ▪ The gel matrix hinders mobility of proteins according to their size and shape. ▪ The gel is commonly polyacrylamide, so separation of proteins via electrophoresis is often called polyacrylamide gel electrophoresis, or PAGE. SDS-PAGE separates proteins by molecular weight SDS – sodium dodecyl sulfate – a detergent SDS micelles bind to proteins and facilitate unfolding. ▪ SDS gives all proteins a uniformly negative charge. ▪ The native shape of proteins does not matter. ▪ The rate of movement will only depend on size: small proteins will move faster. Electrophoresis for protein separation and analysis Electrophoresis: method to visualize and characterize purified proteins Can be used to estimate: ▪ Number of different proteins in a mixture ▪ Degree of protein purity ▪ Isoelectric point of protein ▪ Approximate molecular weight of protein Uses cross-linked polymer polyacrylamide gels Proteins migrate based on charge-to-mass ratio Visualization of proteins: Coomassie blue dye binds to proteins and is visible on gel SDS-PAGE can be used to estimate the molecular weight of a protein Plot of log Mr of marker proteins vs. relative migration during electrophoresis = linear graph ▪ Allows someone to estimate the molecular of other proteins based on the size standards in the gel Isoelectric focusing can be used to determine the pI of a protein Remember: pI = pH where the net charge is zero 2D-electrophoresis: Combining isoelectric focusing and SDS-PAGE Permits resolution of complex protein mixtures of proteins More sensitive than individual methods Aromatic amino acids absorb light The aromatic amino acids absorb light in the UV region. Proteins typically have UV absorbance maxima around 275–280 nm. Tryptophan and tyrosine are the strongest chromophores. For proteins and peptides with known extinction coefficients (or sequences), concentration can be determined by UV- visible spectrophotometry using the Lambert-Beer law: A = cl Specific activity describes the purity of the protein of interest Proteins in a complex mixture often require more than one purification to completely isolate the protein of interest. During purification, determination of the location of the protein of interest can be performed by tracking its behavior. If a protein has a specific function (e.g., binding insulin), the fraction that binds insulin best after each purification step will contain the most of the protein of interest. The total enzyme units in a solution is called the “activity.” The number of enzyme units per mg of total protein is called the “specific activity.” Hypothetical example of protein purification Multi-step process Notice how the total volume is reduced through the process, however... The specific activity is dramatically increased as the protein is purified Protein sequencing It is essential for further biochemical analysis that we know the sequence of the protein we are studying. The actual sequence is generally determined from the DNA sequence, but if unknown… Edman degradation (classical method): ▪ Successive rounds of n-terminal modification, cleavage, and identification ▪ Can be used to identify protein with known sequence Mass spectrometry (modern method): ▪ MALDI-MS and ESI-MS can precisely identify the mass of a peptide, and thus the amino acid sequence ▪ Can be used to determine posttranslational modifications Studying protein structure using proteases Proteases = catalyze hydrolytic cleavage of peptide bonds Edman’s Degradation for protein sequencing MALDI-MS for protein sequencing Some properties of amino acids Notice how the mass difference between two peptides in mass spectrometry can be determined, and... This corresponds to which amino acid is present on one fragment but not on the one that differs by one amino acids based on m/z ratio 160 Chemical synthesis of peptides The Merrifield method: ▪ One end of peptide (C-terminus) = attached to resin in column ▪ Protective chemical groups block unwanted reactions Chemical peptide synthesis is somewhat inefficient This is precisely why small peptides are typically synthesized chemically, but... Proteins of any significant length are synthesized in cells, using cellular machinery (just magnitudes more efficient) Protein sequences as clues to evolutionary relationships Sequences of proteins with identical functions from a wide range of species can be aligned and analyzed for differences. Differences indicate evolutionary divergences. Analysis of multiple protein families can indicate evolutionary relationships between organisms, ultimately the history of life on Earth. https://bmcdevbiol.biomedcentral.com/articles/10.1186/1471-213X-13-18 Chapter 3: Summary In this chapter, we learned about the: ▪ Many biological functions of peptides and proteins ▪ Structures and names of amino acids found in proteins ▪ Ionization properties of amino acids and peptides ▪ Methods for separation and analysis of proteins