Principles of Biochemistry Chapter 1 PDF

Principles of Biochemistry Chapter 1 CH250 Introductory Biochemistry © 2024 James H. Gerlach Grapes and barley are the sources of sugar and natural flavours that are metabolized by live yeast cells to produce alcoholic wine and beer, respectively. Chapter Outline 1....

Principles of Biochemistry Chapter 1 CH250 Introductory Biochemistry © 2024 James H. Gerlach Grapes and barley are the sources of sugar and natural flavours that are metabolized by live yeast cells to produce alcoholic wine and beer, respectively. Chapter Outline 1.1 What Is Biochemistry? 1.2 The Chemical Basis of Life: A Hierarchical Perspective 1.3 Storage and Processing of Genetic Information 1.4 Determinants of Biomolecular Structure and Function 1.1 What Is Biochemistry? Biochemistry Attempts to explain biological processes Molecular level  Cellular level  Organism level  Ecosystem level Interface science Biology Chemistry Biochemical research Mechanistic studies that focus on hypothesis-driven experiments e.g., How do proteins catalyze the synthesis of a complex biomolecule? Relies heavily on the quantitative and statistical analysis of data Rosalind Franklin Helped Decipher the Structure of DNA Rosalind Franklin Biochemists are interested in understanding the structure and function of biological molecules Rosalind Franklin provided the key piece of crystallographic data linking the structure of DNA to its function as the chemical template for genetic inheritance Discovery of Fermentation Eduard Buchner 20 May 1860 – 13 August 1917 German chemist and zymologist 1907 Nobel Prize in Chemistry “For his biochemical researches and his discovery of cell -free fermentation” Produced a cell-free extract of yeast cells and showed that this "press juice" could ferment sugars If glucose, fructose or maltose was added then carbon dioxide was seen to evolve, sometimes for days In World War I Buchner was wounded by shrapnel and died 2 days later at age 57 Büchner Flask and Funnel Used in vacuum filtration Commonly thought to be named for Eduard Buchner Actually named for the industrial chemist Ernst Wilhelm Büchner Yeast Enzymes Are Responsible for Converting Pyruvate into Ethanol and Carbon Dioxide Was Buchner “Lucky” or “Good”? Louis Pasteur had failed to demonstrate cell-free fermentation Majority of scientists believed fermentation was the result of a “vital life force” and Pasteur’s failure reinforced this belief Why did Buchner succeed when others had failed? He used a mixture of quartz sand and diatomaceous earth to grind his yeast Pasteur used ground glass, which made the extract alkaline, inactivating some enzymes He added a 40% sucrose solution as a “preservative” This provided the sugars required for fermentation He used a strain of yeast called Saccharomyces cerevisiae, which was available from breweries in Munich This was a better choice than the strain of yeast used by Pasteur 1.2 The Chemical Basis of Life: A Hierarchical Perspective The Foundation of this Hierarchy Includes Chemical Elements and Functional Groups Chemical Bonding Observed in Biochemistry Common carbon bonds are C–C, C=C, C–H, C=O, C–N, C–S and C–O Molecular Geometry of Carbon Atoms A carbon atom can form a maximum of four single bonds with a tetrahedral arrangement Rotation around a carbon–carbon sigma (σ) single bond is easy, but rotation around a carbon–carbon pi (π) double bond is not possible without breaking the bond Silicon-based Life Forms? Silicon and carbon share many properties Valence of 4, bonds to O, polymerizes Problems with silicon-based life Can only forms bonds with a few types of elements C forms groups with H, O, N, P and S and metals such as Fe, Mg and Zn Has difficulty forming double & triple bonds (Si is larger than C) Silanes are highly reactive with water The silicon hydride (–Si-H) structure reacts with water to yield reactive silanols (-Si-OH) Silicon dioxide is a non-soluble solid Interstellar medium (1998) 84 C-based molecules, only 8 Si-based molecules Trace Elements In addition to the elements listed in Table 1.1, trace elements are used as cofactors in proteins and are required for life These elements are required in smaller (i.e., trace) amounts These elements include: Zinc Iron Manganese Copper Cobalt Essential Ions Play a key role in cell signalling and neurophysiology Include: Calcium Chloride Magnesium Potassium Sodium Elements Essential for Life Functional Groups Play an important role in structure and function of biomolecules Four Major Classes of Small Biomolecules Macromolecules Higher-end structural form of biomolecules Include chemical polymers Proteins – amino acid polymers Nucleic acids – nucleotide polymers Polysaccharides – polymers of monosaccharide molecules (e.g., glucose) Assembling and Disassembling of Macromolecular Polymers Polymers in Macromolecules: Nucleic acids Nucleotides linked by covalent phosphodiester bonds Include DNA and RNA A nucleic acid chain can store information A DNA octamer (8 nucleotides) can have 65,536 (48) different sequence combinations, because each of the 8 positions can have any one of the 4 nucleotides Polymers in Macromolecules: Proteins Amino acids linked by covalent peptide bonds Also known as polypeptides R = different amino acid side chains Polymers in Macromolecules: Polysaccharides Monosaccharides (simple α(1→4) sugars) linked by covalent Amylose (starch) glycosidic bonds e.g., Repeating units of β(1→4) glucose (Glc) or N‐acetylglucosamine Cellulose (GlcNAC) Types of glycosidic bonds β(1→4) are key to the identification and chemical properties of Chitin the polysaccharide Metabolic Pathways Enable cells to coordinate and control complex biochemical processes in response to available energy Function within membrane-bound cells Examples include: Glycolysis and gluconeogenesis (glucose metabolism) Citrate cycle (energy conversion) Fatty acid oxidation and biosynthesis (fatty acid metabolism) Metabolic Pathway Terminology Metabolites Small biomolecules that serve as both reactants and products in biochemical reactions within cells Frequently observed in reactions that are essential in life-sustaining processes Metabolic flux The rate at which reactants and products are interconverted in a metabolic pathway Metabolic Pathway Example: Urea Cycle (Partial) Complex Metabolic Pathways Cellular Structures The Molecular Hierarchy of Structure Organisms A complex organization level that consists of specialized cells Allow multicellular organisms to respond to environmental changes Can adapt to change through signal transduction mechanisms that facilitate cell-cell communication Signal Transduction Human Circulatory System Ecosystems Highest level of hierarchical organization Include co-habitation of different organisms in the same environmental niche Involve a shared use of resources and waste management Ecosystem Examples This is the top rung of the hierarchal ladder of life This is how organisms interact with their environment and each other 1.3 Storage and Processing of Genetic Information In 1952 it was known that DNA alone was sufficient to transmit viral genetic information into infected cells Rosalind Franklin She collected X-ray diffraction data from purified DNA (photo on right) The regular pattern and spacing of the diffraction spots indicated that DNA had a helical structure This helped James Watson and Francis Crick solve the structure of DNA in 1953 Chargaff’s Ratios Erwin Chargaff 1944 – 1952 – used chromatography to quantify bases in DNA Conclusions Base composition generally varies between species Tissues from a species have same base composition Base composition does not change with age, nutritional state or changes in the environment Ratios of certain bases are fixed at 1:1 in all species Chargaff’s Ratios A = T and G = C A+G=T+C Watson and Crick’s Discovery 1953 – Watson and Crick determined that DNA is a double helix Their discovery explained how DNA could pass on genetic material 1962 – Watson, Crick and Wilkins awarded the Nobel Prize in Physiology or Medicine “ It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” – Nature, 1953 Deoxyribonucleotides vs Ribonucleotides Deoxyribonucleotides are monomeric units of DNA that lack an –OH group on the C-2' of the ribose sugar. Ribonucleotides are structurally similar to deoxyribonucleotides, except they contain a –OH at the C-2' position in the ribose sugar. Nucleotide Base Pairs The complimentary base pairs in DNA are: Guanine with cytosine (G:C) Adenine with thymine (A:T) Base pairs are held together by hydrogen bonds 3 bonds in G:C pair 2 bonds in A:T pair Nucleotide Base Pairs in the DNA Double Helix The Central Dogma of Molecular Biology Describes how information is transferred between DNA, RNA and protein Relationship Between DNA and Protein translation translation 1.4 Determinants of Biomolecular Structure and Function An inherent principle in both biology and chemistry is that structure determines function Biological structures are governed by evolutionary processes that impact function This general principle holds true for macromolecules, cells and organisms Proteins need to carry out a variety of biochemical functions that require a vast array of molecular structures The evolutionary driving force creating these diverse protein structures is nucleotide changes in the coding sequences of genes Single Nucleotide Changes in DNA Can Effect the Structure and Function of the Encoded Proteins Proteins acquire a variety of molecular structures through random mutations in DNA Mutations Can occur in germ-line cells Passed from parents to offspring Can result in inherited genetic diseases Can occur in somatic cells Not inherited by the offspring Limited to the individual organism Can result in diseases such as cancer Evolutionary Relationships Can be Represented With a Cladogram Random Mutation and Natural Selection Gene Duplications Can Give Rise to Paralogous Genes Evolutionary Relationships Between Genes Homologous genes (homologs) Share a common ancestor Genes are either homologous or not Percentage similarity NOT percentage homology Orthologous genes (orthologs) Homologs from different species Result from speciation The gene and its main function are conserved Paralogous genes (paralogs) Homologs within a species Results from gene duplication within the species Relationships Between Molecular Structure and Function (1 of 2) Relationships Between Molecular Structure and Function (2 of 2) Physical Biochemistry Chapter 2 CH250 Introductory Biochemistry © 2024 James H. Gerlach The formation of ice crystals inside living organisms can damage membrane integrity and lead to cell death. The yellow mealworm (Tenebrio molitor) larva contains high levels of an antifreeze protein during winter months. Pairs of threonine residues within a repeating sequence of 12 amino acids in the antifreeze protein provide the hydrogen bonding interactions with water molecules that prevent ice crystal growth. Each of the threonine amino acids in the yellow mealworm antifreeze protein has a hydroxyl group that can form hydrogen bonds with H2O at the leading edge of the ice crystal. Chapter Outline 2.1 Energy Conversion in Biological Systems 2.2 Water Is Critical for Life Processes 2.3 Cell Membranes Function as Selective Hydrophobic Barriers 2.1 Energy Conversion in Biological Systems The laws of thermodynamics apply to ALL biological processes Life depends on maintaining a highly ordered steady state called homeostasis, which requires energy Sunlight is the source of energy for almost all biological systems Exergonic and endergonic reactions are often coupled in metabolism The adenylate system (ATP, ADP, AMP) manages most short-term energy needs in biological systems Different Types of Energy Different Types of Work Energy conversion in living systems is required for three types of work Osmotic work Maintains varying [solute] across biological membranes Chemical work Biosynthesis (anabolism) and degradation (catabolism) of organic molecules Mechanical work Muscle contraction in animals Sunlight Is the Source of Energy for Almost All Biological Systems Homeostasis vs. Equilibrium Homeostasis Equilibrium The state of steady internal, Substances transition between physical, and chemical the reactants and products at conditions equal rates, meaning there is no Highly ordered steady state in net change in concentrations terms of temperature, Homeostasis is no longer [biomolecules], etc. maintained Requires energy and delays Macromolecules tend to equilibrium equilibrate to their surroundings Example: living organisms Example: non-living organisms Coupled Oxidation–Reduction (Redox) Reactions Coupled oxidation–reduction (redox) reactions in biological systems Involve the transfer of electrons through a redox circuit The result is provision of energy for chemical work Mnemonics (Memory Aids) for Oxidation and Reduction OIL RIG Oxidation Is Loss of electrons Reduction Is Gain of electrons LEO the lion says GER Loses Electrons, is Oxidized Gains Electrons, is Reduced Photosynthesis and Aerobic Respiration Are the Two Major Energy Conversion Pathways Ice Melting at Room Temperature Is a Process Dominated by the Second Law of Thermodynamics Laws of Thermodynamics (1 of 2) Zeroth law If two thermodynamic systems are each in thermal equilibrium with a third, then they are in thermal equilibrium with each other. First law Energy can neither be created nor destroyed. It can only change forms. In any process in an isolated system, the total energy remains the same. For a thermodynamic cycle, the net heat supplied to the system equals the net work done by the system. Laws of Thermodynamics (2 of 2) Second law The entropy of an isolated system consisting of two regions of space, isolated from one another, each in thermodynamic equilibrium in itself, but not in equilibrium with each other, will, when the isolation that separates the two regions is broken, so that the two regions become able to exchange matter or energy, tend to increase over time, approaching a maximum value when the jointly communicating system reaches thermodynamic equilibrium. Third law As temperature approaches absolute zero, the entropy of a system approaches a constant minimum. Laws of Thermodynamics (Simplified??) Zeroth law There is no heat flow between objects that are the same temperature. First law Heat can neither be created nor destroyed. Second Law "Entropy" is a quantifiable measure of how evenly heat is distributed. Every time heat flows from a hot spot to a cold spot, entropy increases. Every time heat flows from a cold spot to a hot spot, entropy decreases. Third law An ideal engine would convert 100% of heat into useful work only if its exhaust temperature was absolute zero. Therefore 100% efficiency is impossible. Biological Energy Transformations Obey the Laws of Thermodynamics Energy changes in a chemical reaction can be described by three thermodynamic quantities Gibbs energy (G) Enthalpy (H) Entropy (S) For each of these you must know What they represent in biochemical reactions What a positive or negative value indicates What the units of the value are Gibbs Energy G (aka Free Energy) Expresses the amount of energy capable of doing work during a reaction at constant temperature and pressure If Gibbs energy is released, the reaction is exergonic and ΔG has a negative value If Gibbs energy is gained, the reaction is endergonic and ΔG has a positive value The units of ΔG are joules/mole (J mol–1 ) One Joule in Everyday Life Is Approximately The energy required to lift a small apple one metre straight up. (A mass of about 102 g = 1⁄ 9.81 kg) The energy released when that same apple falls one metre to the ground. The energy delivered by a 1-watt solar panel every second. The energy released as heat by a person at rest, every 1/60th of a second. The kinetic energy of a 50 kg human moving very slowly (0.2 m/s or 0.72 km/h). Average walking speed = ~5 km/h Enthalpy H The heat content of the reacting system Reflects the number and kind of chemical bonds in the reactants and the products If heat is released, the reaction is exothermic and ΔH has a negative value If heat is absorbed, the reaction is endothermic and ΔH has a positive value The units of ΔH are joules/mole (J mol–1) Same units as Gibbs energy Entropy S The quantitative expression of the randomness or disorder of a system If the products of a reaction are more complex and more ordered than the reactants, the reaction is said to proceed with a loss in entropy and ΔShas a negative value If the products of a reaction are less complex and more disordered than the reactants, the reaction is said to proceed with a gain in entropy and ΔShas a positive value The units of ΔSare joules/(mole  kelvins) (J mol–1 K–1) Entropy S The quantitative expression of the randomness or disorder of a system If the products of a reaction are more complex and more ordered than the reactants, the reaction is said to proceed with a loss in entropy and ΔShas a negative value If the products of a reaction are less complex and more disordered than the reactants, the reaction is said to proceed with a gain in entropy and ΔShas a positive value The units of ΔSare joules/(mole  kelvins) (J mol–1 K–1) Relationship of the Three Thermodynamic Quantities For constant pressure, volume and temperature ΔH = ΔG + TΔS or ΔG = ΔH – TΔS T is in kelvins (0  C = 273.15 K) ΔH and TΔScan be either negative or positive Every spontaneous reaction has ΔGsystem < 0 All processes that occur spontaneously result in a decrease in the Gibbs energy of the system All processes that occur spontaneously result in an increase in the entropy of the universe (ΔSuniverse > 0) ` Spontaneity of Reactions Exergonic processes (ΔGsystem < 0) Energy-yielding Proceed spontaneously (but may be very slow) C6H12O6 + 6O2  6CO2 + 6H2O + energy Endergonic processes (ΔGsystem > 0) Energy-requiring Cannot proceed spontaneously 6CO2 + 6H2O + energy  C6H12O6 + 6O2 Gibbs energy (kJ/mol) Synthesis of Glucose Oxidation of glucose ΔG = –2880 kJ/mol Synthesis of glucose ΔG = 2880 kJ/mol Changes in Gibbs Energy (ΔG) for the Oxidation and Understanding ΔG Equilibrium Constant (Keq) Ratio of product concentrations to reactant concentrations at equilibrium Moles/litre (mol litre–1) at 25  C A ⇌B then Keq = [B]eq / [A]eq Fructose-6-phosphate ⇌glucose-6-phosphate Keq = [fructose-6-phosphate]eq / [glucose-6-phosphate]eq Keq = 0.5 If the ratio ≠ 0.5, then the reaction will proceed in the direction necessary to make the ratio = 0.5 (Le Chatelier's principle) Gibbs Energy and Chemical Equilibrium Gibbs energy Standard Gibbs Energy Change Standard state used in chemistry Temperature of 298 K (25 °C) Reactants and products initially at 1 M concentrations Or partial pressures of 101.3 kPa (1 atmosphere) if gases Standard Gibbs energy change is ΔG° But [H+] = 1.0 M = pH 0 and [H2O] = 55.5 M Conditions not commonly found in biological systems Standard Gibbs Energy Change Standard state used in biochemistry [H+] = 10 –7 M pH = 7.0 [H2O] = 55.5 M If Mg2+ is present, then the [Mg2+] is constant at 1 mM Standard Gibbs energy change = ΔG°′ Standard equilibrium constant = K′eq When H2O, H+ and/or Mg2+ are reactants or products, their concentrations are incorporated into ΔG°′ and K′eq The Relationship Between ΔG°′ and K′eq Standard Gibbs energy change (ΔG°′ ) Gas constant (R) = 8.314 J mol-1 K-1 Calculation of ΔG°′ (1 of 2) [glucose-1-phosphate] ⇌[glucose-6-phosphate] Calculate the equilibrium constant (K′ eq) Starting conditions [glucose-1-phosphate] = 20 mM [glucose-6-phosphate] = 0 mM Final equilibrium at 25°C at pH 7.0 [glucose-1-phosphate] = 1 mM [glucose-6-phosphate] = 19 mM Calculation of ΔG°′ (2 of 2) ′ [glucose‑ 6‑ phosphate] 19 mM = = = 19 [glucose‑ 1‑ phosphate] 1.0 mM ′ ′ Δ °′ = − ln Δ °′ = − ln Δ °′ = eq eq ′ − ln eq Δ °′ = − 7.3 kJ / mol Calculating ΔG(1 of 2) aA + bB ⇌cC + dD Where a molecules of reactant A combine with b molecules of reactant B to form c molecules of product C and d molecules of product D, then [C]pr[D]pr ∆ = − ln eq + ln [A]pr[B]pr Calculating ΔG(2 of 2) [B]pr [B]eq Where: ∆ = ln − ln ΔG = Gibbs energy change (J/mol) [A]pr [A]eq R= Gas constant (8.314 J/mol·K) [B]pr T = Temperature (K) ∆ = ln − ln ln = Natural logarithm (base e) [A]pr eq [x]pr = Prevailing concentration (mol/L) [B]pr [x]eq = Equilibrium concentration (mol/L) ∆ = − ln + ln Keq = Equilibrium constant (at 298 K) eq [A]pr Gibbs Energy Changes (ΔG) Depend on Temperature and the Concentrations of Reactants and Products A + B⇌ C + D [C]pr[D]pr Δ = Δ °′ + ln [A]pr[B]pr [C]pr[D]pr = mass − action ratio = [A]pr[B]pr Δ = Δ °′ + ln Glycolytic Pathway Reaction 4 (ΔG°′ = +23.8 kJ/mol) ΔG°′ = +23.8 kJ/mol Actual Change in Gibbs Energy (ΔG) for Reaction 4 at 37 °C and Steady-State Concentrations (1 of 4) Fructose-1,6-bisphosphate  Glyceraldehyde-3-phosphate + Dihydroxyacetone phosphate (ΔG°′ = +23.8 kJ/mol) Endergonic – will NOT proceed spontaneously Actual steady-state concentrations of reactant and products in erythrocytes (red blood cells) [Fructose-1,6-bisphosphate] = 0.031 mM = 3.1 x 10–5 M [Dihydroxyacetone phosphate] = 0.138 mM = 1.38 x 10–4 M [Glyceraldehyde-3-phosphate] = 0.019 mM = 1.9 x 10–5 M Actual Change in Gibbs Energy (ΔG) for Reaction 4 at 37 °C and Steady-State Concentrations (2 of 4) Mass action ratio (Q) Q = [B]b[C]c/[A]a Q = [Dihydroxyacetone phosphate][Glyceraldehyde-3-phosphate]/ [Fructose-1,6-bisphosphate] Q = (1.38 x 10–4)(1.9 x 10–5)/(3.1 x 10–5) Q = (2.622 x 10–9)/(3.1 x 10–5) Q = 8.458 x 10–5 Actual Change in Gibbs Energy (ΔG) for Reaction 4 at 37 °C and Steady-State Concentrations (3 of 4) Fructose-1,6-bisphosphate  Glyceraldehyde-3-phosphate + Dihydroxyacetone phosphate (ΔG°′ = +23.8 kJ/mol) ΔG = ΔG°′ + RTln Q ΔG′° = +23.8 kJ/mol = +2.38 x 104 J/mol R= 8.314 J/mol·K T = 37 °C = 310 K Q = 8.458 x 10–5 Actual Change in Gibbs Energy (ΔG) for Reaction 4 at 37 °C and Steady-State Concentrations (4 of 4) ΔG = +2.38 x 104 J/mol + (8.314 J/mol·K)(310 K)(ln 8.458 x 10–5) ΔG = +2.38 x 104 J/mol + (8.314 J/mol·K)(310 K)(–9.378) ΔG = +2.38 x 104 J/mol + –2.42 x 104 J/mol ΔG = –0.04 x 104 J/mol = –0.4 x 103 J/mol ΔG = –0.4 kJ/mol Exergonic – will proceed spontaneously ATP Is a Carrier of Chemical Energy in Living Systems Glutamine Synthesis from Glutamate Is a Two-step Reaction Involving ATP ATP Hydrolysis Can Provide Energy for Protein Conformational Changes Adenylate Kinase Plays a Central Role in Maintaining ATP Levels in the Cell ATP, ADP and AMP Concentrations Vary as a Function of Energy Charge Balanced Flux Through Catabolic and Anabolic Pathways Maintain the Steady State 2.2 Water Is Critical for Life Processes Life as we know it would not be possible without water Life depends on the distinctive chemical properties of water More than 70% of the mass of most cells is water Water plays a central role in biochemical reactions Water has some unusual physical and chemical properties Water is less dense as a solid than as a liquid Water is a liquid over a wide range of temperatures on earth Water is an excellent solvent Many of these properties are due to hydrogen bonding between water molecules The Unique and Unusual Properties of Water (1 of 3) Compound MW Boiling Pt Melting Pt Water (H2O) 18 100  C 0 C Hydrogen Sulphide (H2S) 34 –60  C –84  C Hydrogen Selenide (H2Se) 81 –42  C –64  C Hydrogen Telluride (H2Te) 130 –2  C –49  C The Unique and Unusual Properties of Water (2 of 3) Boiling and melting points Both are unusually high Heat capacity Extremely high heat capacity Useful for heating and cooling Latent heats of fusion and vaporization Both unusually high Ice for cooling, steam for heating The Unique and Unusual Properties of Water (3 of 3) “Universal” solvent More substances dissolve in water than other liquids Surface tension Highest surface tension of any liquid (except mercury) Solid phase Water expands as it cools below 4  C Becomes less dense to 0  C Below 0  C it becomes solid (ice) Surface Tension Water Contains an Oxygen Atom and Two Hydrogen Atoms The Polarity of Water Enables It to Function as Both a Hydrogen-Bond Donor and a Hydrogen-Bond Acceptor In liquid water, water molecules form and break hydrogen bonds approximately every 10 ps. On average, one water molecule is hydrogen bonded to ~3.4 other molecules. Water Density vs. Temperature Ice Is Less Dense Than Liquid Water In ice, each water molecule forms 4 hydrogen bonds (the maximum), creating a crystal lattice. Liquid water Ice Weak Noncovalent Interactions in Biomolecules Biochemical reactions depend on weak interactions 3D structure of proteins and nucleic acids Enzyme–substrate interactions Hormone binding to receptors Stability of DNA double helix Basic types of weak noncovalent interactions Hydrogen bonds Ionic interactions Van der Waals interactions Hydrophobic effects Hydrogen Bonds In Biomolecules Hydrophobic Effects Weak molecular interactions Due to tendency of hydrophobic molecules to pack close together Hydrophobic from greek hydros (water) + phobos (fear) Hydrophilic from greek hydros (water) + philia (friendship) Packing minimizes contact with water molecules Fully-reduced hydrocarbon molecules Nonionic and nonpolar Cannot form hydrogen bonds with water Water molecules form ordered shell around hydrophobic molecules (similar to ordered molecules in surface tension) Ordered Water Molecules and Hydrophobic Effect Introduction of Uncharged Polar and Nonpolar Substances into Water Hydrophobic Effects Are an Important Class of Weak Interactions between Biomolecules Multiple Noncovalent Weak Interactions Are Involved in the Formation of Biomolecular Complexes Osmosis The Osmolarity of a Solution Affects the Size and Shape of Isolated Erythrocytes Ionization of Water A small number of water molecules ionize in solution Ionization of water is a reversible reaction that takes place when two hydrogen-bonded water molecules undergo a bond rearrangement to form a hydronium cation and a hydroxyl anion This reaction is usually written as the ionization of H2O for simplicity and that is the convention your textbook follows H2O ⇌H+ + OH– Ionization of Water Is Expressed by an Equilibrium Constant (1 of 2) Equilibrium constant (Keq) for ionization of water H2O ⇌H+ + OH– Keq = [H+][OH–]/[H2O] Value of ionization equilibrium constant of water at 25  C Determined experimentally from conductivity measurements When pure water is subjected to an electric field H+ ions migrate to the cathode and OH– ions migrate to the anode Keq = 1.8 x 10–16 M Ionization of Water Is Expressed by an Equilibrium Constant (2 of 2) Concentration of water at 25  C Molarity = moles of solute / litres of solution [H2O] = 1000 g/L / 18.015 g/mol [H2O] = 1000 mol / 18.015 L [H2O] = 55.5 M Very large compared to [H+] and [OH–] so change in [H2O] due to ionization is negligible Water Ionization Constant (Kw) Is Calculated from the Equilibrium Constant (1 of 2) To calculate the concentrations of H+ and OH– ions, we rearrange the equilibrium equation (Keq)([H2O]) = [H+][OH–] Substituting for Keq and [H2O] (1.8 x 10–16 M)(55.5 M) = [H+][OH–] = 1.0 x 10–14 M2 Water ionization constant (Kw ) at 25  C Kw = [H+][OH–] Kw = [H+][OH–] = 1.0 x 10–14 M2 Water Ionization Constant (Kw) Is Calculated from the Equilibrium Constant (2 of 2) Neutral pH (no net charge) occurs when [H+] = [OH–] Kw = [H+][OH–] = [H+]2 [H+] = (Kw )1/2 = (1.0 x 10–14 M2)1/2 [H+] = [OH–] = 1.0 x 10–7 M = 10–7 M Acid ionization constant (Ka) Also known as the acid dissociation constant A reflection of the strength of an acid Higher values of Ka indicate a stronger acid HA ⇌H+ + A– Ka = [H+][A–]/[HA] pH Scale pH scale Based on Kw pH definition pH = log (1/[H+]) pH = –log [H+] “p” indicates “negative logarithm of” pOH definition pOH = –log [OH–] pH Is Related to Concentrations of H+ and OH− The Henderson-Hasselbalch Equation Henderson-Hasselbalch equation Relates pH, Ka and buffer concentration Describes the titration curve Lawrence Joseph Henderson (1908) Wrote an equation describing the use of carbonic acid (H2CO3) as a buffer solution Karl Albert Hasselbalch Later re-expressed that formula in logarithmic terms [A–] pH = pKa + log [HA] The Titration Curve of Acetic Acid The Titration Curves of Weak Acids Have Similar Shapes Phosphoric Acid Is a Polyprotic Acid The Carbonic Acid–Bicarbonate Buffer System (1 of 2) The buffer system maintains blood pH at 7.40 In this biological buffering system, the weak acid is carbonic acid (H2CO3) and the conjugate base is bicarbonate (HCO3−) H2CO3 ⇌H+ + HCO3− Although the pKa for this reaction at 37 °C is only 6.1, the carbonic acid–bicarbonate conjugate pair can function as the primary buffering system in blood because it is in equilibrium with three other processes that together adjust H2CO3 and HCO3− levels The Carbonic Acid–Bicarbonate Buffer System (2 of 2) One of these processes is a reversible reaction catalyzed by the enzyme carbonic anhydrase The other two processes are exchange of CO2(aq) in the blood with atmospheric CO2(g) in the air spaces of the lungs, and the excretion of HCO3− (urine) or retention of HCO3− (blood) through the kidneys Respiratory Regulation of Blood pH The body regulates the respiratory rate using chemoreceptors, which primarily use CO2 as a signal. Peripheral blood sensors are found in the walls of the aorta and carotid arteries. These sensors signal the brain to provide immediate adjustments to the respiratory rate if CO2 levels rise or fall. The Bicarbonate Buffering System Plays a Key Role in Maintaining Blood pH Levels (pH 7.35 – 7.45) 2.3 Cell Membranes Function as Selective Hydrophobic Barriers Membranes Create the boundary between a cell and its environment Interface allowing exchange of nutrients and wastes Structure of biological membranes Hydrophobic barrier containing amphipathic lipid molecules Amphipathic from Greek amphi (both) + pathos (suffering) Both hydrophilic and hydrophobic properties Phospholipids Most abundant lipid class found in membranes Polar charged head group (hydrophilic) Long nonpolar hydrocarbon tails (hydrophobic) Biological Membranes Phospholipids Are Amphipathic Biomolecules Amphipathic Phospholipids Can Associate into Four Different Types of Complexes When Mixed with Water Side View of a Phospholipid Bilayer Liposomes Can Function as Drug-Delivery Systems Nucleic Acid Structure and Function Chapter 3 CH250 Introductory Biochemistry © 2024 James H. Gerlach Chapter Outline 3.1 Structure of DNA and RNA 3.2 Genomics: The Study of Genomes 3.3 Methods in Nucleic Acid Biochemistry 3.1 Structure of DNA and RNA Nucleotides – The Building Blocks of DNA and RNA DNA Structure 1 nm = 10 Å 1 Å = 10–10 m Hydrogen-Bonding Interactions Hold Base Pairs Together Two Antiparallel Strands Can Form Base Pairs Nucleotide Base Stacking Stabilizes the DNA Double Helix The A, B and Z Conformations Are Three Arrangements of the DNA Double Helix Reversible Denaturation and Annealing (Renaturation) of DNA Melting Temperature Is Influenced by Strand Length and Ionic Strength Supercoiled DNA Is Generated by Twisting of the Double -Stranded Helix DNA Supercoiling Is Induced During Transcription by RNA Polymerase Topological Strain Caused by Supercoiling Is Relieved by DNA Type I Topoisomerases Topoisomerase II Enzymes Change Supercoiling in DNA Molecules Schematic Representations of Duplex, Hairpin, Bulge and Loop RNA Secondary Structures The Ribozyme RNaseP Contains Several Examples of RNA Secondary Structure in RNA G–U base pairing (blue arrows) The Structure of tRNA Is Important for Its Function Structures of the Modified Nucleosides Found in tRNA Base Pairing of Nucleosides Found in tRNA Histone Proteins Bind to DNA in a Sequence- Independent Manner The lac Repressor Protein Dimer Binds to DNA Sequence Specifically The lac Repressor Protein Dimers Form DNA Loops The crystal structures of the lac repressor show that it forms a bent structure, with all four of the DNA-binding portions pointing in one direction. Based on this structure, researchers have proposed that when all four subunits bind at the same time, the DNA is twisted into a small loop. 3.2 Genomics: The Study of Genomes Genome A genome is the genetic material of an organism. It consists of DNA (or RNA in RNA viruses like SARS-CoV-2). It includes both the genes (the coding regions), the noncoding DNA and the genetic material of the mitochondria and chloroplasts. The Size of the Genomic DNA for a Variety of Organisms Condensation of Eukaryotic DNA Into Chromosomes Telomeres Protect the Ends of Chromosomes From Degradation Genes Are Regions of DNA that Contain a Coding Sequence for Functional Biomolecules The Organization of a Typical Eukaryotic Gene Bioinformatics Is Used in Biochemistry to Discover the Function of an Unknown Gene Bioinformatic Analysis Is Ushering in the Age of Precision Medicine The Spread of Infectious Disease Can be Mapped Using DNA Sequencing and Bioinformatics Tracking COVID-19: SARS-CoV-2 Coronavirus Mutations Genomic Epidemiology of SARS-CoV-2 3.3 Methods in Nucleic Acid Biochemistry Steps in DNA Cloning 1. Cutting target DNA at precise locations. Sequence-specific endonucleases (restriction endonucleases) provide the necessary molecular scissors. 2. Selecting a small carrier molecule of DNA capable of self- replication. These DNAs are called cloning vectors (a vector is a delivery agent). They are typically plasmids or viral DNAs. 3. Joining two DNA fragments covalently. The enzyme DNA ligase links the cloning vector and the DNA to be cloned. Composite DNA molecules comprising covalently linked segments from two or more sources are called recombinant DNAs. Steps in DNA Cloning 4. Moving recombinant DNA from the test tube to a host cell that will provide the enzymatic machinery for DNA replication. 5. Selecting or identifying host cells that contain recombinant DNA. Plasmid Vectors Facilitate Propagation of Recombinant Molecules in Antibiotic-Resistant Bacteria Restriction Endonucleases Recognize Palindromic Sequences in DNA and Cleave Double-Stranded DNA Genomic DNA Cloning Is Based on Methods Collectively Called Recombinant DNA Technology Protein-Coding Sequences Can be Cloned Using mRNA That Is Converted to cDNA The Chain-Termination Method Can be Used to Determine the Sequence of a Region of DNA The Polymerase Chain Reaction (PCR) Kary Mullis (December 28, 1944 – August 7, 2019) Invented the polymerase chain reaction (PCR) technique. Shared the 1993 Nobel Prize in Chemistry with Michael Smith. In 1983, Mullis was working for Cetus Corporation as a chemist. While driving his Honda Civic on Highway 128 from San Francisco to Mendocino, he had the idea to use a pair of oligonucleotide primers to bracket a desired DNA sequence and then copy it using DNA polymerase. In his Nobel Prize lecture, he remarked that the success did not make up for his girlfriend breaking up with him. “I was sagging as I walked out to my little silver Honda Civic. Neither [assistant] Fred, empty Beck's bottles, nor the sweet smell of the dawn of the age of PCR could replace Jenny. I was lonesome.” He received a $10,000 bonus from Cetus for the invention, who later sold the patent rights to Hoffmann La-Roche for $300,000,000. The Polymerase Chain Reaction Is an in vitro Method of DNA Amplification The PCR Amplification Cycle Consists of Three Temperature Phases Gene Expression Analysis Can Be Performed Using a Gene Array (Microarray) RNA-seq Is an Unbiased Transcriptome Analysis Method A Hypothetical Experiment Comparing the Results of a Differential Gene Expression Study CRISPR-Cas9: An RNA-Guided DNA Targeting Tool Discovered in the 1990s CRISPR = Clustered Regularly Interspersed Short Palindromic Repeats Cas9 = CRISPR-associated protein 9 CRISPR sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote A form of adaptive immunity based on specific recognition of bacteriophage DNA by complementary RNA that was transcribed Cas9 is an enzyme that uses CRISPR sequences to recognize and cleave DNA that are complementary to the CRISPR sequence CRISPR-Cas9 that can be used to edit genes within organisms The CRISPR-Cas9 System Consists of an RNA–Protein Complex That Recognizes Specific DNA Sequences An Application of the CRISPR-Cas9 System Is Genome Editing of Target DNA