General Biochemistry II: Complete Study Guide on Biological Catalysis, Metabolic Pathways, Bioenergetics, and Electrophoresis

General Principles of Metabolism and Biological Catalysis

• Metabolism Definition and Fundamental Architecture:
  • Life relies on a vast, interconnected network of biochemical transformations termed metabolism.
  • Metabolism comprises two primary opposing arms:
    • Anabolism: Biosynthetic processes that construct complex cellular structures and components from simpler precursors.
    • Catabolism: Degradative processes that break down complex nutrient molecules to extract biologically usable energy and generate simple metabolic wastes.
  • Metabolic operations encompass essential cellular functions:
    • Building, repairing, and restoring damaged tissues.
    • Converting food and structural nutrients into bioactive energy units (such as adenosine triphosphate, $ATP$).
    • Disposing of cellular metabolic waste products.

• Thermodynamic Energy Barriers and Catalytic Necessity:

  • A central feature of cellular chemistry is that the vast majority of vital metabolic reactions do not proceed spontaneously at significant rates under physiological conditions.
  • Reactant molecules exist in stable ground states that require substantial activation energy ($E_a$) inputs to reach the transition state needed for chemical conversion.
  • Living systems overcome this energy barrier using biological catalysts called enzymes.
  • Catalysis: The acceleration of a chemical reaction rate by a substance that undergoes no permanent chemical change itself at the end of the process.

• In Vitro vs. In Vivo Hydrolysis Comparison:

  • In Vitro (Laboratory Conditions): Complete non-enzymatic hydrolysis of a standard protein requires boiling in highly corrosive $20\%\,HCl$ for approximately $24\,\text{hours}$.
  • In Vivo (Biological Conditions): Enzymatic protein digestion in biological systems is completed in under $4\,\text{hours}$ under extremely gentle conditions ($ext{pH}\approx 7.4$, temperature $37^\circ\text{C}$).

Structure, Chemical Nature, and Components of Enzymes

• Chemical Properties of Enzymes:

  • Enzymes are specialized biological catalysts (biocatalysts) that exhibit high reaction specificity and catalytic efficiency.
  • Chemically, almost all enzymes are globular proteins composed of linear amino acid chains joined by peptide bonds, organized into precise tertiary or quaternary structural conformations.
  • Their molecular weight ($MW$) ranges from $10{,}000\,\text{Dalton}$ to $2{,}000{,}000\,\text{Dalton}$.
  • The primary amino acid sequence dictates the three-dimensional folding pattern, which directly establishes catalytic activity.

• The Active Site and Catalytic Triad Structure:

  • The Active Site is a specialized pocket, cleft, or crevice on the enzyme surface formed by the folding of the tertiary protein structure.
  • It creates a unique hydrophobic microenvironment that optimizes substrate binding and chemical transformation.
  • Contains two distinct functional categories of amino acid residues:
    • Binding Residues: Recognize, orient, and hold the substrate molecule via non-covalent interactions.
    • Catalytic Residues: Directly participate in the bond-breaking and bond-forming chemical steps.
  • Example (Serine Protease): $\alpha$-Chymotrypsin is a pancreatic digestive enzyme that cleaves dietary protein peptide bonds in the small intestine. Its active site relies on a catalytic triad of three specific amino acids brought together in 3D space: Aspartate 102 ($ ext{Asp}\,102$), Histidine 57 ($ ext{His}\,57$), and Serine 195 ($ ext{Ser}\,195$).

• Simple vs. Conjugated Enzymes:

  • Simple Enzymes: Consist entirely of polypeptide protein chains with no additional non-protein chemical components (e.g., Pancreatic Ribonuclease).
  • Conjugated Enzymes: Require non-protein helper components to attain full catalytic activity.

• Enzyme System Components and Definitions:

  • Apoenzyme: The inactive, purely protein component of a conjugated enzyme system.
  • Cofactor: The non-protein molecule or ion required for catalytic activity.
  • Holoenzyme: The complete, biochemically active enzyme-cofactor complex:         $\text{Holoenzyme} = \text{Apoenzyme} + \text{Cofactor}$
  • Coenzymes: Organic, non-protein helper molecules that are heat-stable, dialyzable, and loosely associated with the apoenzyme, often functioning as transient chemical group carriers.
  • Prosthetic Groups: Organic cofactors that are tightly or covalently bound to the apoenzyme matrix and do not dissociate during catalysis (e.g., Biotin covalently attached to pyruvate carboxylase).
  • Metal Ion Activators / Inorganic Cofactors: Essential transition metal cations ($Fe^{2+}$, $Fe^{3+}$, $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$, $Cu^{2+}$) that provide structural stability, orient substrates, or stabilize ionic charge buildup during transition states.

• Major Coenzymes and Transferred Entities:

  • Thiamine Pyrophosphate ($TPP$): Transfers aldehydes.
  • Tetrahydrofolate ($THF$): Transfers one-carbon functional units.
  • Pyridoxal Phosphate ($PLP$): Transfers amino groups.
  • Nicotinamide Adenine Dinucleotide ($NAD^+ / NADH$): Transfers hydrogen atoms and electrons.
  • Flavin Adenine Dinucleotide ($FAD / FADH_2$): Transfers hydrogen atoms and electrons.
  • Coenzyme A ($CoA\text{-}SH$): Transfers acyl groups.
  • Biocytin: Transfers carbon dioxide ($CO_2$).
  • $3'$-Deoxyadenosylcobalamin (Coenzyme $B_{12}$): Transfers hydrogen atoms and alkyl groups.

Enzyme Classification, Nomenclature, and History

• Historical Discovery and Milestones:

  • The word enzyme originally referred to "in yeast" or fermentation processes.
  • 1926: James B. Sumner crystallized the enzyme urease from jack beans, establishing that enzymes are protein molecules. Urease was the first enzyme ever crystallized.
  • Non-protein catalysts also exist:
    • Ribozymes: Catalytic $RNA$ molecules capable of cleaving nucleic acid chains.
    • Abzymes: Catalytic antibodies engineered to possess specific enzyme-like catalytic activity.
    • Polysaccharide Catalysts: Cyclodextrins and related carbohydrates exhibiting catalytic properties.

• Types of Enzyme Specificity:

  • Absolute Specificity: The enzyme catalyzes a reaction for only one unique substrate molecule (e.g., Urease acting exclusively on urea).
  • Group Specificity: The enzyme acts only on molecules sharing a specific functional group (e.g., Hexokinase transferring phosphate to various hexose sugars).
  • Linkage Specificity: The enzyme targets a specific covalent bond type regardless of the molecular structure surrounding it (e.g., Trypsin hydrolyzing peptide linkages).
  • Stereochemical Specificity: The enzyme distinguishes between optical D- and L- stereoisomers, acting on only one configuration (e.g., L-amino acid oxidase).

• Enzyme Nomenclature and Enzyme Commission (EC) Classes:

  • Traditional historical names (e.g., pepsin, trypsin, rennin) give no structural or reaction information.
  • Modern standard systematic naming appends the suffix "-ase" to the substrate or catalyzed reaction.
  • The International Union of Biochemistry and Molecular Biology (IUBMB) established numerical Enzyme Commission ($EC$) classifications categorized into six major structural classes based on reaction type (OTHLIL):
    • EC 1: Oxidoreductases: Catalyze oxidation-reduction reactions where electrons are transferred between donor and acceptor molecules (e.g., Lactate Dehydrogenase: $\text{Pyruvate} + \text{NADH} + \text{H}^+ \rightleftharpoons \text{L-Lactate} + \text{NAD}^+$).
    • EC 2: Transferases: Catalyze the transfer of specific functional groups (such as phosphate, methyl, or amino groups) from a donor to an acceptor molecule (e.g., Hexokinase; Aspartate Carbamoyltransferase).
    • EC 3: Hydrolases: Catalyze the cleavage of bonds via the addition of water (hydrolysis) (e.g., Trypsin [classified as EC 3]; Glucose-6-phosphatase: $\text{Glucose-6-phosphate} + \text{H}_2\text{O} \rightarrow \text{Glucose} + \text{P}_i$).
    • EC 4: Lyases: Catalyze the addition of groups across double bonds or the non-hydrolytic removal of groups to form double bonds (e.g., Fumarate Hydratase/Fumarase: $\text{L-Malate} \rightleftharpoons \text{Fumarate} + \text{H}_2\text{O}$).
    • EC 5: Isomerases: Catalyze structural, geometric, or conformational rearrangements within a single molecule to produce an isomeric form (e.g., Alanine Racemase: $\text{L-Alanine} \rightleftharpoons \text{D-Alanine}$).
    • EC 6: Ligases: Catalyze the joining of two distinct molecules driven by the hydrolysis of ATP or another nucleoside triphosphate ($NTP$) (e.g., Glutamine Synthetase: $\text{Glutamate} + \text{NH}_3 + \text{ATP} \rightarrow \text{Glutamine} + \text{ADP} + \text{P}_i$).

Catalytic Mechanisms and Binding Models

• Enzyme-Substrate Binding Models:
  • Emil Fischer's Lock and Key Model (1894): Posits a rigid, pre-formed active site into which a complementary substrate fits precisely, like a key into a lock. This static model fails to account for enzyme flexibility, transition state stabilization, or allosteric control.
  • Daniel Koshland's Induced Fit Model (1958): Posits a flexible, dynamic active site. Binding of the substrate induces a conformational change in the enzyme, wrapping the catalytic site tightly around the substrate to align functional groups into optimal position for transition state stabilization.

• Stages of the Catalytic Cycle:

  1. Substrate ($S$) enters and binds the enzyme active site to form the reversible Enzyme-Substrate complex ($ES$):         $E + S \rightleftharpoons ES$
  2. The $ES$ complex undergoes chemical rearrangement through the transition state to form the Enzyme-Product complex ($EP$):         $ES \rightleftharpoons EP$
  3. Product ($P$) dissociates from the active site, freeing the enzyme ($E$) unchanged to bind a new substrate molecule:         $EP \rightleftharpoons E + P$

• Molecular Acceleration Mechanisms:

  • Proximity Effect: Organizes and concentrates substrate molecules within the active site volume, creating local substrate concentrations far higher than in bulk solution.
  • Orientation Effect: Holds reactive bonds in precise spatial alignment and geometric angles required for optimal bond breaking.
  • Acid-Base Catalysts: Active site amino acid side chains act as specific proton donors (acids) or proton acceptors (bases).
  • Electrostatic Catalysts: Charged side chains (e.g., Lysine, Arginine, Aspartate, Glutamate) or bound metal ions form ionic interactions that stabilize polar transition state charge build-up.
  • Covalent Catalytic Actions: Active site nucleophiles form transient, unstable covalent bonds with the substrate, creating an alternative low-energy reaction pathway.
  • Metal Ion Catalysts: Bound cations coordinate substrates, polarize bonds, shield negative charges, and facilitate redox electron transfers independent of solution pH.

• Thermodynamics of Activation Energy:

  • Enzymes accelerate chemical reaction rates exclusively by lowering the activation energy barrier ($E_a$) required to reach the transition state.
  • They do not alter the overall free energy change (Standard Free Energy, $\Delta G$) of the reaction.
  • They do not alter the final chemical equilibrium constant ($K_{eq}$); instead, they increase the rate of both the forward and reverse reactions equally.

Enzyme Kinetics and Quantitative Activity

• Units of Enzyme Activity and Turnover Rates:

  • Enzyme Activity: The rate of catalytic conversion of substrate to product under defined conditions of temperature, pH, and substrate concentration.
  • International Unit ($IU$ or $U$): Defined as the amount of enzyme that catalyzes the conversion of $1\,\mu\text{mol}$ of substrate per minute ($1\,\mu\text{mol\,min}^{-1}$) under specified optimal conditions.
  • Turnover Number ($k_{cat}$): The maximum number of substrate molecules converted into product per active site per second when the enzyme is fully saturated with substrate:         $k_{cat} = \frac{V_{max}}{E_T}$
  • Representative Turnover Rates ($k_{cat}$ in $\text{mol product\,s}^{-1}\text{\,mol enzyme}^{-1}$):
    • Carbonic Anhydrase: $600{,}000\,\text{s}^{-1}$
    • Catalase: $93{,}000\,\text{s}^{-1}$
    • $\beta$-Galactosidase: $200\,\text{s}^{-1}$
    • Chymotrypsin: $100\,\text{s}^{-1}$
    • Tyrosinase: $1\,\text{s}^{-1}$

• Factors Regulating Reaction Rates:

  • Substrate Concentration ($[S]$): At low $[S]$, reaction rate is directly proportional to $[S]$ (first-order kinetics). As $[S]$ increases, active sites become saturated and rate approaches a maximum constant velocity $V_{max}$ (zero-order kinetics).
  • Enzyme Concentration ($[E]$): Reaction velocity increases linearly with increasing enzyme concentration, provided substrate is present in excess.
  • Product Concentration: Accumulation of reaction products causes feedback inhibition or drives the reverse reaction.
  • Temperature: Increasing temperature boosts kinetic energy and collision frequency. However, exceeding the optimum temperature (typically $40^\circ\text{C} - 50^\circ\text{C}$ in animals) causes thermal denaturation of protein structure. Thermophilic bacteria enzymes remain active up to $100^\circ\text{C}$. The Temperature Coefficient ($Q_{10}$) measures rate change per $10^\circ\text{C}$ rise ($Q_{10} \approx 2$ for stable biological processes).
  • pH: Enzyme activity forms a characteristic bell-shaped curve when plotted against pH due to ionization changes of active site amino acid side chains and substrate functional groups. Optimum pH values vary by biological niche:
    • Pepsin (gastric fluid): $\text{pH}\approx 2$
    • Amylase (saliva/pancreas): $\text{pH}\approx 7$
    • Trypsin (duodenum): $\text{pH}\approx 8.5$
    • Alkaline Phosphatase: $\text{pH} > 9$
  • Time: Product formation increases over time, but initial linear rates decay over prolonged incubation due to progressive thermal denaturation and substrate depletion.

• Michaelis-Menten Kinetic Model:

  • The Michaelis-Menten equation relates initial velocity ($v_1$ or $v_0$) to substrate concentration ($[S]$):         $v = \frac{V_{max}[S]}{K_m + [S]}$
  • Derivation Assumptions: $[S] \gg [E]$; the formation of $ES$ complex reaches a steady state; the chemical conversion step ($ES \rightarrow E + P$) is the rate-limiting step.
  • Mathematical Conditions:
    • When $[S] \ll K_m$: $K_m + [S] \approx K_m$, simplifying the equation to $v = \frac{V_{max}}{K_m}[S]$ (First-order kinetics).
    • When $[S] \gg K_m$: $K_m + [S] \approx [S]$, simplifying the equation to $v = V_{max}$ (Zero-order kinetics).
    • When $[S] = K_m$: $v = \frac{V_{max}[S]}{2[S]} = \frac{V_{max}}{2}$. Thus, the Michaelis Constant ($K_m$) is experimentally defined as the substrate concentration required to reach half of the maximum velocity ($V_{max}/2$).
  • Significance of $K_m$: $K_m$ reflects enzyme affinity for a substrate. A lower $K_m$ value indicates higher binding affinity (less substrate is needed to reach half-saturation), whereas a higher $K_m$ indicates lower affinity.

• Lineweaver-Burk Double Reciprocal Plot:

  • Taking the reciprocal of the Michaelis-Menten equation yields the linear equation ($y = mx + c$):         $\frac{1}{v} = \left(\frac{K_m}{V_{max}}\right) \cdot \frac{1}{[S]} + \frac{1}{V_{max}}$
  • Plot Parameters:
    • Y-axis: $\frac{1}{v}$
    • X-axis: $\frac{1}{[S]}$
    • Y-intercept: $\frac{1}{V_{max}}$
    • X-intercept: $-\frac{1}{K_m}$
    • Slope: $\frac{K_m}{V_{max}}$

• Reversible Enzyme Inhibition and Regulatory Controls:
  • Competitive Inhibition: Inhibitor structural analog competes directly with substrate for the free enzyme active site. Increases apparent $K_m$ (lowers affinity); $V_{max}$ remains unchanged (can be overcome by increasing $[S]$).
  • Non-Competitive Inhibition: Inhibitor binds allosterically to a distinct site on both $E$ and $ES$. Lowers $V_{max}$; $K_m$ remains unchanged.
  • Uncompetitive Inhibition: Inhibitor binds strictly to the $ES$ complex, preventing product formation. Lowers both $V_{max}$ and apparent $K_m$.
  • Control Mechanisms: Regulation of enzyme levels occurs via gene expression/induction, repression, covalent modification, zymogen proenzyme cleavage/activation, and end-product feedback inhibition.

Carbohydrate Catabolism and Glycolysis

• Properties and Structure of Glucose:

  • Glucose is a six-carbon hexose, an aldohexose sugar, and exists predominantly as a six-membered pyranose ring in solution.

• Glycolytic Pathway Overview:

  • Glycolysis (the Embden-Meyerhof-Parnas pathway) is a cytosolic sequence of 10 enzyme-catalyzed reactions that converts one molecule of glucose into two molecules of pyruvate.
  • It operates independently of molecular oxygen ($O_2$) under both aerobic and anaerobic conditions.

• Key Enzymatic Reactions and Regulation:

  • Step 1 (Phosphorylation): Glucose is phosphorylated to glucose-6-phosphate by Hexokinase (classified as $EC\,2$, transferase), consuming 1 molecule of $ATP$.
    • Function: Traps glucose inside the cytoplasm by adding a charged phosphate group, preventing efflux across the plasma membrane and initializing glucose degradation.
  • Key Regulatory Enzyme: Phosphofructokinase-1 (PFK-1) catalyzes the rate-limiting, irreversible phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate.
  • High-Energy Intermediate: Phosphoenolpyruvate (PEP) contains a high-energy phosphate ester bond hydrolyzable to release energy.

• Energy Yield and Energetics:

  • Gross Yield: $4\,\text{ATP} + 2\,\text{NADH}$
  • Energy Investment: $2\,\text{ATP}$ consumed (Steps 1 and 3)
  • Net Yield per Glucose: $2\,\text{ATP} + 2\,\text{NADH} + 2\,\text{Pyruvate}$
  • Substrate-level phosphorylation generates $ATP$ directly in the cytosol without requiring the electron transport chain or oxygen.
  • Cells dependent solely on glycolysis for energy include mature erythrocytes (red blood cells, which lack mitochondria).

Pyruvate Fates and Cellular Respiration

• Anaerobic Fates of Pyruvate (Fermentations):

  • Under anaerobic or hypoxic conditions, cells must regenerate the electron acceptor $NAD^+$ from accumulated $NADH$ to keep glycolysis functioning.
  • Lactic Acid Fermentation: Pyruvate is reduced directly to L-lactate by Lactate Dehydrogenase ($EC\,1$), consuming $NADH + H^+$:         $\text{Pyruvate} + \text{NADH} + \text{H}^+ \rightleftharpoons \text{L-Lactate} + \text{NAD}^+$$\Delta G'^circ = -25.1\,\text{kJ\,mol}^{-1}$
    • Occurs in vigorously contracting skeletal muscle, erythrocytes, and lactic acid bacteria.
  • Ethanol Fermentation: Pyruvate is decarboxylated to acetaldehyde by Pyruvate Decarboxylase (requiring $TPP$ and $Mg^{2+}$ cofactors), releasing $CO_2$. Acetaldehyde is then reduced to ethanol by Alcohol Dehydrogenase, consuming $NADH$ and regenerating $NAD^+$. Occurs in yeast cells.

• Aerobic Fate of Pyruvate (Pyruvate Oxidation):

  • Under aerobic conditions, pyruvate enters the mitochondrial matrix for complete oxidative breakdown.
  • It undergoes irreversible oxidative decarboxylation into Acetyl-CoA catalyzed by the multi-enzyme Pyruvate Dehydrogenase Complex (PDH) (comprising $E_1$, $E_2$, and $E_3$ enzymes):         $\text{Pyruvate} + \text{CoA\text{-}SH} + \text{NAD}^+ \xrightarrow{\text{PDH}} \text{Acetyl-CoA} + \text{CO}_2 + \text{NADH} + \text{H}^+$$\Delta G'^circ = -33.4\,\text{kJ\,mol}^{-1}$
  • Required Cofactors: Thiamine Pyrophosphate ($TPP$), Lipoic Acid/Lipoate, Flavin Adenine Dinucleotide ($FAD$), Coenzyme A ($CoA\text{-}SH$), and Nicotinamide Adenine Dinucleotide ($NAD^+$).

The Citric Acid Cycle (TCA / Krebs Cycle)

• Discovery and Cellular Location:
  • Discovered by Sir Hans Krebs in 1937 (awarded the Nobel Prize in Physiology or Medicine in 1953).
  • Also known as the Tricarboxylic Acid ($TCA$) cycle or Krebs cycle.
  • Occurs entirely within the mitochondrial matrix in eukaryotic cells (with the exception of succinate dehydrogenase, which is membrane-bound).

• The Eight Reaction Steps of the TCA Cycle:

  1. Citrate Formation: Condensation of 2-carbon Acetyl-CoA with 4-carbon Oxaloacetate and $H_2O$ to form 6-carbon Citrate, catalyzed by Citrate Synthase:         $\text{Acetyl-CoA} + \text{Oxaloacetate} + \text{H}_2\text{O} \rightarrow \text{Citrate} + \text{CoA\text{-}SH}$$\Delta G'^circ = -32.2\,\text{kJ\,mol}^{-1}$
  2. Isomerization to Isocitrate: Dehydration of Citrate to cis-Aconitate followed by rehydration to Isocitrate, catalyzed by Aconitase:         $\text{Citrate} \rightleftharpoons \text{cis-Aconitate} + \text{H}_2\text{O} \rightleftharpoons \text{Isocitrate}$$\Delta G'^circ = 13.3\,\text{kJ\,mol}^{-1}$
  3. First Oxidative Decarboxylation: Oxidation of Isocitrate to $\alpha$-Ketoglutarate ($5\text{C}$), releasing $CO_2$ and generating $NADH$, catalyzed by Isocitrate Dehydrogenase via an oxalosuccinate intermediate (requires $Mn^{2+}$):         $\text{Isocitrate} + \text{NAD(P)}^+ \rightarrow \alpha\text{-Ketoglutarate} + \text{CO}_2 + \text{NAD(P)H} + \text{H}^+$
  4. Second Oxidative Decarboxylation: Multienzyme complex oxidative decarboxylation of $\alpha$-Ketoglutarate to 4-carbon Succinyl-CoA, releasing $CO_2$ and generating $NADH$, catalyzed by the $\alpha$-Ketoglutarate Dehydrogenase Complex:         $\alpha\text{-Ketoglutarate} + \text{CoA\text{-}SH} + \text{NAD}^+ \rightarrow \text{Succinyl-CoA} + \text{CO}_2 + \text{NADH}$$\Delta G'^circ = -33.5\,\text{kJ\,mol}^{-1}$
  5. Substrate-Level Phosphorylation: Hydrolysis of the high-energy thioester bond of Succinyl-CoA to form Succinate, coupled to the phosphorylation of $GDP$ to $GTP$ (or $ADP$ to $ATP$), catalyzed by Succinyl-CoA Synthetase:         $\text{Succinyl-CoA} + \text{GDP} + \text{P}_i \rightleftharpoons \text{Succinate} + \text{GTP} + \text{CoA\text{-}SH}$$\Delta G'^circ = -2.9\,\text{kJ\,mol}^{-1}$
  6. Dehydrogenation to Fumarate: Oxidation of Succinate to Fumarate by Succinate Dehydrogenase, transferring $2\text{H}^+$ and $2e^-$ to $FAD$ to form $FADH_2$:         $\text{Succinate} + \text{FAD} \rightleftharpoons \text{Fumarate} + \text{FADH}_2$$\Delta G'^circ = 0\,\text{kJ\,mol}^{-1}$
     • Special Note: Succinate dehydrogenase is the only $TCA$ cycle enzyme tightly bound to the inner mitochondrial membrane (functioning as Complex II in the electron transport chain).
  7. Hydration to L-Malate: Stereospecific hydration of Fumarate across its double bond to form L-Malate via a carbanion transition state, catalyzed by Fumarase:         $\text{Fumarate} + \text{H}_2\text{O} \rightleftharpoons \text{L-Malate}$$\Delta G'^circ = -3.8\,\text{kJ\,mol}^{-1}$
  8. Dehydrogenation to Oxaloacetate: Oxidation of L-Malate back to Oxaloacetate, generating $NADH + H^+$, catalyzed by L-Malate Dehydrogenase:         $\text{L-Malate} + \text{NAD}^+ \rightleftharpoons \text{Oxaloacetate} + \text{NADH} + \text{H}^+$$\Delta G'^circ = 29.7\,\text{kJ\,mol}^{-1}$

• Stoichiometry of One TCA Cycle Turn:

  • $\text{Acetyl-CoA} + 3\,\text{NAD}^+ + \text{FAD} + \text{GDP} + \text{P}_i + 2\,\text{H}_2\text{O} \rightarrow \text{CoA\text{-}SH} + 2\,\text{CO}_2 + 3\,\text{NADH} + \text{FADH}_2 + \text{GTP} + 3\,\text{H}^+$
  • Every single turn releases $2\,\text{molecules of CO}_2$, $3\,\text{NADH}$, $1\,\text{FADH}_2$, and $1\,\text{GTP/ATP}$.

• Anaplerotic (Replenishing) Reactions:

  • $TCA$ cycle intermediates serve as biosynthetic precursors (amphibolic pathway). As intermediates are siphoned off, they must be replenished by anaplerotic reactions to maintain constant cycle concentration:
    • Pyruvate Carboxylase: $\text{Pyruvate} + \text{HCO}_3^- + \text{ATP} \rightleftharpoons \text{Oxaloacetate} + \text{ADP} + \text{P}_i$ (Liver, kidney).
    • PEP Carboxykinase: $\text{Phosphoenolpyruvate} + \text{CO}_2 + \text{GDP} \rightleftharpoons \text{Oxaloacetate} + \text{GTP}$ (Heart, skeletal muscle).
    • PEP Carboxylase: $\text{Phosphoenolpyruvate} + \text{HCO}_3^- \rightleftharpoons \text{Oxaloacetate} + \text{P}_i$ (Plants, yeast, bacteria).
    • Malic Enzyme: $\text{Pyruvate} + \text{HCO}_3^- + \text{NAD(P)H} \rightleftharpoons \text{Malate} + \text{NAD(P)}^+$ (Widely distributed in eukaryotes and prokaryotes).

• Regulation of PDH and the TCA Cycle:

  • Pyruvate Dehydrogenase Complex: Inhibited by $ATP$, acetyl-CoA, $NADH$, and fatty acids. Activated by $AMP$, $CoA\text{-}SH$, $NAD^+$, and $Ca^{2+}$.
  • Citrate Synthase: Inhibited by $NADH$, succinyl-CoA, citrate, and $ATP$. Activated by $ADP$.
  • Isocitrate Dehydrogenase: Inhibited by $ATP$. Activated by $ADP$ and $Ca^{2+}$.
  • $\alpha$-Ketoglutarate Dehydrogenase: Inhibited by succinyl-CoA and $NADH$. Activated by $Ca^{2+}$.

The Glyoxylate Cycle

• Bypass Pathway in Plants, Bacteria, and Fungi:
  • Animals cannot perform net conversion of fatty acids (acetyl-CoA) into carbohydrates (glucose) because $PDH$ is irreversible and $TCA$ decarboxylation steps lose carbon as $CO_2$.
  • Plants (in specialized organelles called glyoxysomes), fungi, and bacteria utilize the Glyoxylate Cycle to skip the two $CO_2$-releasing decarboxylation steps of the $TCA$ cycle.

• Key Bypass Steps and Specific Enzymes:
  1. Acetyl-CoA condenses with Oxaloacetate to form Citrate, which isomerizes to Isocitrate.
  2. Isocitrate Lyase cleaves Isocitrate directly into 4-carbon Succinate and 2-carbon Glyoxylate.
  3. Malate Synthase condenses Glyoxylate with a second molecule of Acetyl-CoA to form L-Malate.
  4. L-Malate is oxidized to Oxaloacetate to continue the cycle.
  5. Succinate produced in the glyoxysome is exported to the mitochondrion, converted to malate via $TCA$ enzymes, and transported to the cytosol for glucose production via gluconeogenesis.

Bioenergetics, Redox Potentials, and Oxidative Phosphorylation

• Redox Potentials of Respiratory Chain Components:

  • Oxidation involves loss of electrons, loss of hydrogen, or addition of oxygen ($ext{Fe}^{2+} \rightarrow \text{Fe}^{3+} + e^-$).
  • Redox potential ($E^\circ$) measures a molecule's electron affinity:
    • Lowest Redox Potential: Hydrogen ($H_2$), $NAD^+/NADH$.
    • Highest Redox Potential: Coenzyme Q, Cytochrome $c$, Cytochrome $a/a_3$, Oxygen ($O_2$).

• Electron Transport Chain and Respiration Coupling:

  • Aerobic dehydrogenases contain $FMN$ or $FAD$ prosthetic groups.
  • Most respiratory chain complexes are tightly attached to the inner mitochondrial membrane, whereas Coenzyme Q (ubiquinone) is a lipid-soluble mobile carrier.
  • When reducing equivalents enter via $NADH$, the P:O ratio (moles of $ATP$ synthesized per atom of oxygen reduced) is $3:1$.
  • The rate of tissue respiration is regulated primarily by intracellular concentration of $[ADP]$ (acceptor control).

• Specific Respiratory Inhibitors and Uncouplers:

  • Cyanide ($CN^-$): Inhibits electron transport and oxidative phosphorylation at Site III (Complex IV / Cytochrome $c$ oxidase).
  • Oligomycin: Inhibits ATP synthesis by binding directly to the $F_0$ subunit of mitochondrial ATP synthase.
  • 2,4-Dinitrophenol (DNP): Functions as an uncoupler of oxidative phosphorylation. DNP makes the inner mitochondrial membrane permeable to protons ($H^+$), dissipating the proton motive force without blocking electron transport, thereby halting $ATP$ synthesis while releasing energy as heat.
  • Arsenate: Inhibits $ATP$ production by competing with inorganic phosphate ($P_i$).
  • Direct Driving Energy Source: The electrochemical proton gradient ($H^+$ concentration difference) across the inner mitochondrial membrane drives $ATP$ synthesis via $F_1/F_0$ ATP Synthase.

Fundamental Principles and Physics of Electrophoresis

• Etymology and Definition:

  • Derived from Greek: Electro (electricity) + Phoresis (transport, movement, or separation).
  • Electrophoresis: A physical analytical method involving the migration and separation of charged molecules, ions, or colloidal particles in a conducting liquid or gel matrix under the influence of an applied electric field.

• Electrophoresis vs. Electrolysis:

  • Electrolysis: A non-spontaneous chemical reaction driven by electric current to completely decompose electrolytes; cations undergo reduction at the negative cathode, while anions undergo oxidation at the positive anode to generate new chemical products.
  • Electrophoresis: An incomplete form of electrolysis where the electric field is turned off before molecules reach the physical electrodes, preserving the chemical identity of separated biological compounds for analytical analysis.

• Governing Physical Equations and Kinetics:

  • Potential Gradient ($E$):$E = \frac{V}{d}$(Where $V$ is applied voltage in volts, and $d$ is the distance between electrodes in meters).
  • Driving Electrostatic Force ($F$):$F = E \times q$(Where $q$ is the net molecule charge in Coulombs). This force drives charged ions toward the oppositely charged electrode.
  • Retarding Frictional Drag Force ($F_{friction}$):$F_{friction} = f \times v$(Where $f$ is the frictional coefficient and $v$ is migration velocity). $f$ depends on hydrodynamic particle size, shape, gel pore density, and buffer viscosity.
  • Migration Velocity ($v$):$v = \frac{E \times q}{f}$
  • Electrophoretic Mobility ($m$): The ratio of ion migration velocity to electric field strength:         $m = \frac{v}{E} = \frac{q}{f}$

• Amino Acid Zwitterions and Isoelectric Point ($pI$):

  • Amino acids exist as dipolar internal salts called zwitterions around neutral pH, carrying balanced positive ($ ext{-NH}_3^+$) and negative ($ ext{-COO}^-$) functional groups resulting in a net charge of zero.
  • Isoelectric Point ($pI$): The specific pH value unique to each molecule at which its net overall electrical charge is exactly zero (number of positive charges = number of negative charges).
  • Cataphoresis: The electrophoretic movement of positively charged ions (cations) toward the negative cathode.
  • Anaphoresis: The electrophoretic movement of negatively charged ions (anions) toward the positive anode.

• Historical Timeline of Advances:
  • 1807 (Ruess): First demonstrated electrophoretic movement using clay particles dispersed in water migrating toward the anode under an electric field.
  • 1860s: Optical methods developed to detect moving boundaries in liquids.
  • 1937 (Arne Tiselius): Built the U-shaped glass tube apparatus for moving boundary electrophoresis in free solution without supporting media, using Schlieren optics to detect refractive index boundaries (described in "A New Apparatus for Electrophoretic Analysis of Colloidal Mixtures").
  • 1940s–1950s: Advent of Zone Electrophoresis utilizing solid supporting media (Consden - Paper Electrophoresis 1952; Smithies - Starch Gel Electrophoresis 1955; Kohn - Cellulose Acetate Electrophoresis 1958; Svensson - Isoelectric Focusing 1961).
  • 1970s–1990s: Capillary and microchip methods developed (Everaerts - Capillary Isotachophoresis 1970; Jörgenson & Lukacs - Capillary Electrophoresis 1981; Terabe - Micellar Electrokinetic Chromatography 1984; Hjerte'n - Capillary IEF 1985; Manz - Microchip Electrophoresis 1992).

Key Factors Influencing Electrophoretic Separation

• Sample Properties:

  • Net Charge and Charge Density ($q$): Higher net charge increases electrostatic force and migration rate toward the opposite electrode.
  • Molecular Mass and Hydrodynamic Size: The gel acts as a molecular sieve; smaller molecules navigate matrix pores easily and move faster than larger molecules.
  • Conformational Shape: Compact, folded globular molecules encounter less frictional resistance ($f$) than elongated, fibrous unfolded structures of identical molecular weight.
  • Relative Hydrophobicity: Hydrophobic proteins tend to aggregate or stick non-specifically to the stationary gel matrix, causing poor resolution or band smearing.

• Electric Field Characteristics:

  • Field Strength / Voltage ($V$): Higher voltage increases migration velocity; however, excessive voltage generates Joule heating in the buffer solution, causing thermal convection currents, band distortion, and sample denaturation.

• Buffer System Properties:

  • Buffer pH: Dictates the net charge of amphoteric molecules like proteins:
    • If $\text{pH} > pI$, the protein loses protons, becoming negatively charged (anion) and migrating toward the anode (+).
    • If $\text{pH} < pI$, the protein gains protons, becoming positively charged (cation) and migrating toward the cathode (-).
    • If $\text{pH} = pI$, net charge is zero, and all electrophoretic migration stops completely.
  • Ionic Strength: High buffer ionic strength increases current and heat generation while slowing sample migration; low ionic strength reduces resolution.

• Gel Matrix and Electroendosmosis:

  • Pore Size: Acts as a molecular sieve; controlled by matrix polymer concentration.
  • Electroendosmosis (EEO): Osmotic movement of liquid buffer relative to a fixed charged matrix (caused by ionized carboxyl or sulfate groups on gel surfaces), which retards or distorts sample migration bands.

Classification, Techniques, and Applications of Electrophoresis

• General Classification of Electrophoretic Systems:

  • Free Electrophoresis (Solution Systems): Performed in aqueous electrolyte solutions in U-tubes without solid supporting media; susceptible to thermal convection mixing and diffusional broadening.
  • Zone Electrophoresis (Carrier Systems): Performed on solid, porous, or gel supporting media to prevent convection and diffusional mixing, separating samples into distinct isolated zones.

• Major Zone Electrophoresis Techniques:

  • Paper Electrophoresis: Uses paper filter strips (low adsorption capacity) to separate small charged molecules like amino acids.
  • Cellulose Acetate Electrophoresis (Kohn 1958): Uses chemically pure, non-hydrophilic cellulose acetate membranes that absorb minimal buffer, permitting rapid, high-resolution separation of serum proteins and polysaccharides.
  • Agarose Gel Electrophoresis: Uses large-pore polysaccharide gels purified from seaweed; optimal for separating large nucleic acid fragments ($200$ to $50{,}000\,\text{base pairs}$). Has low resolving power for small protein differences.
  • Polyacrylamide Gel Electrophoresis (PAGE): Formed by polymerizing monomeric acrylamide cross-linked with $N,N'$-methylenebisacrylamide (bis-acrylamide). The combined total concentration determines pore size. Has high resolving power, separating DNA fragments differing by a single base pair ($<500\,\text{bp}$).
  • SDS-PAGE (Sodium Dodecyl Sulfate-PAGE): Anionic detergent SDS denatures proteins into linear rod-like polypeptides and coats them with a uniform negative charge proportional to chain length. SDS-PAGE separates proteins strictly by polypeptide chain molecular weight ($MW$), eliminating native shape and charge variations.
  • Native PAGE: Conducted under non-denaturing conditions without detergents; retains protein folding, native quaternary structure complexes, and biological enzymatic activity.
  • Western Blotting: Extends SDS-PAGE by electrotransferring separated protein bands onto a nitrocellulose or $PVDF$ membrane (electroblotting), followed by specific primary/secondary antibody binding and chemiluminescent detection for protein identification and quantification.
  • Isoelectric Focusing (IEF): Uses carrier ampholytes to establish a continuous pH gradient in a gel matrix under an electric field. Proteins migrate until reaching the exact pH equal to their $pI$ where net charge is zero. Extremely sensitive; resolves proteins differing by a few hundredths of a pH unit. Requires specialized equipment.
  • Two-Dimensional Gel Electrophoresis (2D-PAGE): Combines Isoelectric Focusing in the first dimension (separating by $pI$) with SDS-PAGE in the second dimension (separating by $MW$), achieving high-resolution profiling of complex proteomes.
  • Capillary Electrophoresis (CE): High-voltage separation carried out inside narrow-bore fused silica capillaries ($25-100\,\mu\text{m}$ inner diameter); offers rapid separation speed and high efficiency using minute sample volumes.
  • Isotachophoresis (ITP): Sample components migrate at equal uniform speeds in discrete sequential zones sandwiched between a high-mobility leading electrolyte and a low-mobility terminating electrolyte.
  • Immunoelectrophoresis: Combines electrophoretic separation with antibody immunoprecipitation to identify specific proteins (such as immunoglobulins) via precipitin arcs.

• Comprehensive Practical Applications:

  • Molecular Biology & Genetics: PCR product verification, restriction fragment analysis, DNA fingerprinting, genomic sequencing, and $RNA$ integrity checks.
  • Clinical Diagnostics & Medicine: Serum Protein Electrophoresis (SPE) to diagnose multiple myeloma, liver cirrhosis, and nephrotic syndrome; Hemoglobin Electrophoresis to screen for sickle cell anemia and thalassemia; detection of oligoclonal bands in multiple sclerosis.
  • Forensic Science & Criminology: DNA profiling from crime scene evidence; analysis of organic gunshot additives (ethylcentralite, nitroglycerin) via Micellar Electrokinetic Capillary Chromatography; illicit drug testing.
  • Pharmaceutical Quality Control & Biotechnology: Purity testing of recombinant protein therapeutics, monoclonal antibodies, and vaccine potency/concentration.
  • Food & Agriculture: Allergen detection, ingredient authenticity verification, and genetic marker identification in crop and livestock breeding.