Biochemistry and Thermodynamics Lecture Flashcards

Bioenergetics of Glucose Phosphorylation

• Standard Free-Energy Changes ($\Delta G^\circ$) for Coupled Reactions:     * Reaction 1: $\text{Glucose} + P_i \rightarrow \text{Glucose-6-phosphate}$ has a $\Delta G^\circ = 13.8\,kJ/mol$.     * Reaction 2: $\text{ADP} + P_i \rightarrow \text{ATP}$ has a $\Delta G^\circ = 30.5\,kJ/mol$.

• Calculating the Free-Energy Change for the Net Reaction:     * Target reaction: $\text{Glucose} + \text{ATP} \rightarrow \text{Glucose-6-phosphate} + \text{ADP}$.     * This is the sum of (Reaction 1) and the reverse of (Reaction 2).     * Reverse Reaction 2: $\text{ATP} \rightarrow \text{ADP} + P_i$ ($\Delta G^\circ = -30.5\,kJ/mol$).     * Sum: $13.8 + (-30.5) = -16.7\,kJ/mol$.

Bioenergetics and Equilibrium Calculations

• ATP Synthesis Thermodynamics:     * Given $K_{eq}$ for the breakdown of ATP to ADP is $2.22 \times 10^5\,M$ at $25^{\circ}C$.     * To calculate $\Delta G^\circ$ for the synthesis of ATP from ADP and $P_i$:         * $\Delta G^\circ = -RT \ln(K_{eq})$.         * The $K_{eq}$ for synthesis is the reciprocal of the breakdown $K_{eq}$: $\frac{1}{2.22 \times 10^5}$.

• Gibbs Free Energy Symbols and Definitions:     * $\Delta G$: Actual change in Gibbs free energy based on current concentrations.     * $\Delta G^\circ$: Standard free energy change at standard temperature ($25^{\circ}C$) and pressure ($1\,atm$ or $101.3\,kPa$).     * $\Delta G'^\circ$: Biochemical standard free energy change at $\text{pH } 7.0$.

• Equilibrium Constant of Glucose Phosphorylation:     * Reaction: $\text{Glucose} + P_i \rightarrow \text{Glucose-6-phosphate}$.     * Givens: $\Delta G'^\circ = 13.8\,kJ/mol$ (endergonic, unfavored, not spontaneous).     * Temperature: $37^{\circ}C$ ($310\,K$).     * Gas Constant ($R$): $8.314\,J \cdot K^{-1} \cdot mol^{-1}$.     * Relationship: $\Delta G'^\circ = -RT \ln(K_{eq})$.

Physiological Concentrations and Reaction Drivers

• Direct Phosphorylation in Rat Hepatocytes:     * Physiological [Glucose] and [$P_i$]: $4.8\,mM$ ($0.0048\,M$).     * Determining potential [Glucose-6-phosphate] through direct phosphorylation: This step is generally considered unreasonable for glucose catabolism due to the unfavorable thermodynamic barrier (\Delta G'^\circ > 0).

• Substrate Increase as a Driver:     * One could theoretically drive the reaction by significantly increasing substrate concentration [Glucose], but biological ranges are limited.

• Coupling to ATP Hydrolysis:     * Is glucose phosphorylation feasible when coupled to ATP? Yes, because the net $\Delta G$ becomes negative.     * Typical cellular values: [Glucose-6-phosphate] = $250\,\mu M$; [ATP] = $3.38\,mM$; [ADP] = $1.32\,mM$.

Pop Quiz Review: Ramachandran Plots and Amino Acids

• Question 1: Which angle describes the X-axis (horizontal) on a Ramachandran plot?     * Answer: Phi ($\phi$).

• Question 2: Identify which labeled point (A, B, C, or D) is most likely a glycine.     * Answer: Glycine can occupy regions of the plot that are restricted for other amino acids due to its lack of a bulky side chain.

• Question 3: How many classes of hairpin turns were described?     * Answer: 4.

• Questions 4 and 5: Identify the amino acid and its $pK_a$.     * Answer: Histidine; $pK_a = 6.0$.

Principles of Biological Redox Reactions

• Fundamental Concepts:     * Oxidation: Loss of electrons ($OIL$ - Oxidation Is Loss).     * Reduction: Gain of electrons ($RIG$ - Reduction Is Gain).     * These processes always occur concurrently as redox reactions.     * Oxidant (Oxidizing Agent): Accepts electrons and is reduced.     * Reductant (Reducing Agent): Donates electrons and is oxidized.

• Biological Context:     * Energy generation stems from electron movement between species with differing electron affinities.     * Electromotive Force (emf): The force resulting from electron transfer that can perform biological work.     * Foods (e.g., glucose) are reduced compounds. As they are oxidized, electrons flow toward species with higher affinity, like $O_2$, which is exergonic.     * Mitochondrial Coupling: Electron flow is coupled to the creation of a proton ($H^+$) gradient across the inner membrane, resulting in proton-motive force used by ATP synthase.

Carbon Oxidation States and Electronegativity

• Electronegativity Order: H < C < S < N < O.

• Valence Accounting:     * Carbon ($C$) has 4 valence electrons and can form up to 4 covalent bonds.     * The more electronegative atom in a bond "owns" the shared electrons.

• Oxidation States of Carbon:     * Methane ($CH_4$): Carbon is more electronegative than Hydrogen; Carbon "owns" all 8 bonding electrons. Formal oxidation state: $-4$ (Most reduced).     * Ethane ($(CH_3)_2$): $C-C$ bond electrons are shared equally. Each Carbon "owns" 7 electrons. Formal oxidation state: $-3$.     * Carbon Dioxide ($CO_2$): Carbon "owns" zero electrons as Oxygen is more electronegative. Formal oxidation state: $+4$ (Most oxidized).

• General Rule: Loss of electrons or addition of Oxygen indicates Carbon oxidation.

Electron Transfer Mechanisms and Reduction Potentials

• Four Ways to Transfer Electrons:     1. Directly as electrons: e.g., $Fe^{2+} + Cu^{2+} \rightarrow Fe^{3+} + Cu^+$.     2. As Hydrogen atoms: A hydrogen atom = $1\,e^- + 1\,H^+$. Pair: $A / AH_2$.     3. As Hydride ions ($:H^-$): Contains 2 electrons (common in $NAD$-linked reactions).     4. Direct combination with Oxygen: Oxygen is covalently incorporated into the product (e.g., $R-CH_3 + \frac{1}{2}O_2 \rightarrow R-CH_2-OH$).

• Reducing Equivalent: A single electron equivalent transferred in a redox reaction.

• Reduction Potential ($E$): A measure clearly in Volts ($V$) of the affinity of an electron acceptor for electrons.     * Standard Reference: The hydrogen half-cell (H-electrode) is set to $0.0\,V$ for the reaction $H^+ + e^- \rightarrow \frac{1}{2}H_2$ at $\text{pH } 0$.     * Biochemical Standard Potential ($E'^\circ$): Measured at $\text{pH } 7.0$, $298\,K$, $1\,M$ solutes, and $101.3\,kPa$ gas pressure.     * Electrons flow from lower $E'^\circ$ to higher $E'^\circ$. Higher affinity = more positive voltage.

• Relationship to Free Energy:     * $\Delta E'^\circ = E'^\circ(\text{acceptor}) - E'^\circ(\text{donor})$.     * $\Delta G'^\circ = -nF \Delta E'^\circ$.     * $n$ = number of electrons transferred.     * $F$ (Faraday Constant) = $96,480\,J/V \cdot mol$.

• Nernst Equation: used to calculate actual reduction potential under non-standard concentrations:     * $E = E'^\circ + \frac{RT}{nF} \ln\left(\frac{[\text{electron acceptor}]}{[\text{electron donor}]}\right)$.

Specialized Electron Carriers: NAD, NADP, FAD, and FMN

• Nicotinamide Adenine Dinucleotide (NAD/NADP):     * Synthesized from Niacin (Vitamin $B_3$).     * Soluble, dissociates from enzymes after each cycle.     * Transfer involves a hydride ion ($:H^-$), representing $2\,e^-$ simultaneously.     * NAD+/NADH: High cellular ratio (\text{NAD}^+ > \text{NADH}); functions in catabolism (oxidation); predominantly mitochondrial.     * NADP+/NADPH: Low cellular ratio (\text{NADPH} > \text{NADP}^+); functions in anabolism (reductive biosyntheses); predominantly cytosolic.

• Flavin Coenzymes (FAD/FMN):     * FAD: Flavin Adenine Dinucleotide; FMN: Flavin Mononucleotide.     * The prosthetic groups of flavoproteins; tightly bound and do not dissociate.     * Can transfer electrons one at a time (forming a semi-quinone) or two at a time ($FADH_2 / FMNH_2$).     * $E'^\circ$ varies based on the protein environment (Range: $-0.40\,V$ to $+0.06\,V$; free FAD is $-0.219\,V$).

Respiratory Chain and Metabolic Redox Problems

• Case 1: NADH Oxidation in Respiratory Chain:     * $\text{NADH} + H^+ + \frac{1}{2}O_2 \rightleftharpoons H_2O + \text{NAD}^+$.     * Half-reactions:         * $\text{NAD}^+ + H^+ + 2e^- \rightarrow \text{NADH}$ ($E'^\circ = -0.320\,V$).         * $\frac{1}{2}O_2 + 2H^+ + 2e^- \rightarrow H_2O$ ($E'^\circ = +0.816\,V$).     * $\Delta E'^\circ = 0.816 - (-0.320) = 1.136\,V$.     * $\Delta G'^\circ = -(2)(96480)(1.136) \approx -219.2\,kJ/mol$.     * If ATP synthesis requires $52\,kJ/mol$, this reaction could theoretically generate $4$ molecules of ATP ($219.2 / 52 = 4.2$).

• Case 2: Pyruvate to Lactate Conversion:     * $E'^\circ$ for Pyruvate/Lactate = $-0.185\,V$.     * $E'^\circ$ for $NAD^+ / NADH$ = $-0.320\,V$.     * Redox pair with greatest tendency to lose electrons: $NAD^+ / NADH$ (more negative potential).     * Stronger oxidizing agent: Pyruvate (it has a higher/less negative potential, meaning it has a higher affinity to accept electrons).     * $\Delta E'^\circ(\text{Pyruvate} \rightarrow \text{Lactate}) = -0.185 - (-0.320) = 0.135\,V$.     * $\Delta G'^\circ = -(2)(96480)(0.135) \approx -26.1\,kJ/mol$.

• Case 3: Acetaldehyde to Ethanol:     * Reaction: $\text{Acetaldehyde} + \text{NADH} + H^+ \rightarrow \text{Ethanol} + \text{NAD}^+$.     * Givens: [Acetaldehyde] = [NADH] = $1.0\,M$; [Ethanol] = [$NAD^+$] = $0.10\,M$.     * Standard potentials: Acetaldehyde/Ethanol = $-0.197\,V$; $NAD^+ / NADH$ = $-0.320\,V$.

Protein Folding and Thermodynamics

• Folding Process Steps:     1. Folding.     2. Cofactor binding.     3. Covalent modification.     4. Translocation.     5. Assembly into multi-subunit complexes.

• Thermodynamic Principles:     * Proteins fold spontaneously only when \Delta G < 0.     * Equation: $\Delta G = \Delta H - T \Delta S$.     * Entropy ($\Delta S$): Folding is an "ordering" of the polypeptide (negative $\Delta S$), but it is driven by the release of ordered water molecules (the solvated shell) into the bulk solvent, causing a net increase in system disorder (positive $\Delta S$). Therefore, folding is primarily entropically driven.     * Enthalpy ($\Delta H$): Generally close to zero. Favorable non-covalent interactions (hydrophobic, polar, charged) are often balanced by the loss of hydrogen bonds with water.

Models and Pathways of Protein Folding

• Levinthal’s Paradox: A 100-residue protein would take $20\,billion$ years to fold if it sampled every conformation randomly; thus, folding must follow specific pathways.

• Folding Pathways and Energy Landscapes:     * Local free energy minima: Intermediate states where the protein temporarily resides.     * Global free energy minimum: The native, functional state.     * Energy Wells: Proteins want to reach the lowest possible energy state. If a protein gets "trapped" in a false local minimum (biological "energy well"), it requires help to escape.

• Current Folding Models:     1. Hierarchical: Local secondary structures (helices, sheets) form first, then interact to form tertiary structures.     2. Hydrophobic Collapse: Polypeptide rapidly collapses to hide hydrophobic groups from water, forming a "Molten Globule."

• Computational Status: Humans cannot yet perfectly predict folding from primary sequence algorithmically, though AI (Deep Learning/AlphaFold) has seen success, albeit without us fully understanding the AI’s internal logic.

Molecular Chaperone Systems (Hsp70 and Hsp60)

• General Purpose: Heat Shock Proteins (Hsp) are upregulated during thermal stress to rescue denatured proteins, though they assist in normal folding as well.

• Hsp70 Family (e.g., bacterial DnaK):     * Binds to hydrophobic patches on proteins emerging from the ribosome.     * Prevents premature folding during membrane translocation.     * Mechanism:         1. Hsp70 complexed with ATP binds the polypeptide, aided by Hsp40 (DnaJ).         2. ATP hydrolysis to ADP causes a conformational change that locks Hsp70 to the protein.         3. ADP/Hsp40 dissociate.         4. New ATP binding releases the polypeptide.

• Hsp60 Family (e.g., bacterial GroEL/ES):     * Acts on fully synthesized polypeptides.     * Structure: A barrel-like complex.     * Mechanism:         1. Polypeptide binds the hydrophobic rim.         2. 7 ATP molecules bind, opening the chamber and pulling the protein inside.         3. A protein cap (GroES) closes the chamber, providing a protected environment.         4. ATP hydrolysis causes the cap to dissociate and release the protein.     * Capacity: Max polypeptide size is approximately $57\,kDa$ ($\sim 520$ residues).

Protein Refinement, Modification, and Denaturation

• Folding Enzymes:     1. Protein Disulfide Isomerases (PDI): Shuffles/corrects disulfide bonds between Cys residues.     2. Peptide Prolyl Cis-Trans Isomerase (PPI): Catalyzes interconversion of proline isomers.     3. Proteases: Cleave signal sequences or pro-sequences to activate proteins or target them for secretion.

• Denaturation (Loss of Fold and Function):     * Thermal Denaturation: Heat disrupts weak non-covalent interactions.     * Melting Temperature ($T_m$): The midpoint of the cooperative unfolding transition.     * Chemical Denaturants: Urea, detergents, and organic solvents (acetone, alcohol) disrupt the hydrophobic core.     * pH Effects: Change the net charge, causing electrostatic repulsion.     * Reducing Agents: Mercaptoethanol reduces disulfide cross-links.

• Estimation of Protein Weight:     * The approximate weight of a protein in Daltons is calculated as the Number of Residues $\times 110$.     * Example: A 100-residue protein is roughly $11,000\,Da$.