Biochemistry and Metabolic Pathways Flashcards

Enzyme Kinetics, Regulation, and Inhibition

• Enzymes are biological catalysts that accelerate reaction rates without being altered or consumed in the process ($E + S \rightleftharpoons ES \rightarrow E + P$).

• Enzyme regulation is essential for coordinating metabolic networks and prioritizing cellular requirements.

• Inhibitors diminish the velocity of enzyme-catalyzed reactions; many clinical drugs operate as enzyme inhibitors.

Energy Changes During Chemical Reactions

• An energy barrier separates reactants (initial state $A$) and products (final state $B$).

• The free energy of activation ($E_a$ or $\Delta G^\ddagger$) is the energy difference between the reactants and the high-energy transition state ($T^*$).

• Reactant molecules must possess sufficient kinetic energy to overcome the transition state barrier; the overall reaction rate is determined by the number of molecules possessing this energy threshold.

• Lowering the free energy of activation increases the fraction of molecules reaching the transition state, thereby accelerating reaction velocity ($\downarrow E_a \rightarrow \uparrow \text{rate}$).

• Enzymes provide an alternative reaction pathway with a lower free energy of activation, but they do NOT alter the overall free energy change of the reaction ($\Delta G = G_{\text{reactants}} - G_{\text{products}}$).

Chemistry of the Active Site

• The active site is a flexible molecular machine that binds substrate ($S$) and stabilizes it in the transition state ($T^*$).

• It enhances the probability of transition state formation through precise spatial positioning, electrostatic stabilization, and brief covalent interactions.

Factors Affecting Reaction Velocity

• Substrate Concentration ($[S]$):

  • Initial reaction velocity ($v_0$) increases with substrate concentration until maximal velocity ($V_{\max}$) is reached.

  • At $V_{\max}$, all enzyme active sites are fully saturated with substrate; further increases in $[S]$ produce no additional velocity increase.

• Temperature:

  • Human enzymes function optimally at physiological body temperature ($37\,^{\circ}\text{C}$).

  • Excessive heat causes thermal inactivation and denaturation of enzyme secondary and tertiary structure.

• pH:

  • Enzymes display peak catalytic efficiency at specific pH optima reflecting their tissue environment:

  • Pepsin (stomach acid): Optimal pH $\approx 1.5 - 2.0$

  • Trypsin (small intestine): Optimal pH $\approx 6.0 - 8.0$

  • Alkaline phosphatase (alkaline secretions): Optimal pH $\approx 9.0 - 10.5$

Michaelis-Menten Kinetics

• Reaction Model:   $E + S \underset{k_{-1}}{\overset{k_1}{\rightleftharpoons}} ES \xrightarrow{k_2} E + P$

• Michaelis-Menten Equation:   $v_0 = \frac{V_{\max} [S]}{K_m + [S]}$

• Michaelis Constant ($K_m$):

  • Defined mathematically as $K_m = \frac{k_{-1} + k_2}{k_1}$.

  • Expressed in units of concentration (e.g., $mM$ or $\mu M$).

  • Equals the exact substrate concentration at which initial reaction velocity reaches half of maximal velocity ($v_0 = \frac{1}{2} V_{\max}$).

• Substrate Affinity and $K_m$:

  • A small $K_m$ reflects high substrate affinity; low $[S]$ is required to achieve $\frac{1}{2} V_{\max}$.

  • A large $K_m$ reflects low substrate affinity; high $[S]$ is required to achieve $\frac{1}{2} V_{\max}$.

• Kinetic Curves:

  • Enzymes following classical Michaelis-Menten kinetics display a hyperbolic velocity curve.

  • Allosteric enzymes display a sigmoidal velocity curve, reflecting subunit cooperativity.

Lineweaver-Burk Plot

• Linear transformation of the Michaelis-Menten equation using double reciprocal values ($\frac{1}{v_0}$ vs $\frac{1}{[S]}$):   $\frac{1}{v_0} = \left(\frac{K_m}{V_{\max}}\right) \frac{1}{[S]} + \frac{1}{V_{\max}}$

• Intercept on the x-axis equals $-\frac{1}{K_m}$.

• Intercept on the y-axis equals $\frac{1}{V_{\max}}$.

• Slope of the line equals $\frac{K_m}{V_{\max}}$.

Types of Enzyme Inhibition

• Competitive Inhibition:

  • Inhibitor structurally resembles the substrate and competes for binding at the active site.

  • Effect on $V_{\max}$: Unchanged; at high $[S]$, substrate outcompetes the inhibitor to reach full $V_{\max}$.

  • Effect on $K_m$: Increased; higher $[S]$ is required to achieve $\frac{1}{2} V_{\max}$.

  • Examples:

  • Malonate structurally mimics succinate and competitively inhibits succinate dehydrogenase.

- Statin drugs (e.g., Lovastatin) competitively inhibit HMG-CoA reductase to decrease cholesterol synthesis.

• Noncompetitive Inhibition:

  • Inhibitor binds to an allosteric/distinct site on free enzyme ($E$) or enzyme-substrate complex ($ES$).

  • Effect on $V_{\max}$: Decreased; high $[S]$ cannot overcome the inhibition.

  • Effect on $K_m$: Unchanged; substrate affinity for the active site is unaffected.

  • Example: Lead poisoning causes irreversible noncompetitive inhibition of ferrochelatase and ALA dehydratase in heme biosynthesis.

• Uncompetitive Inhibition:

  • Inhibitor binds exclusively to the $ES$ complex.

  • Decreases both $V_{\max}$ and $K_m$; cannot be reversed by increasing substrate concentration.

Regulation of Enzyme Activity

• Allosteric Regulation:

  • Allosteric enzymes contain multiple subunits and catalyze rate-limiting steps.

  • Effectors bind non-covalently at allosteric sites ("other sites").

  • Positive effectors increase catalytic activity or substrate affinity.

  • Negative effectors decrease catalytic activity or substrate affinity.

  • Homotropic effectors: Substrate itself acts as the allosteric effector (displays cooperativity, producing a sigmoidal curve).

  • Heterotropic effectors: Effector is a distinct molecule from the substrate (e.g., feedback inhibition where end product $E$ inhibits step $A \rightarrow B$).

• Covalent Modification:

  • Addition or removal of phosphate groups on specific Serine (Ser), Threonine (Thr), or Tyrosine (Tyr) residues.

  • Protein Kinases catalyze phosphorylation using ATP ($\text{Protein} + \text{ATP} \rightarrow \text{Protein-P} + \text{ADP}$).

  • Protein Phosphatases catalyze dephosphorylation ($\text{Protein-P} + \text{H}_2\text{O} \rightarrow \text{Protein} + \text{P}_i$).

• Induction and Repression:

  • Transcriptional or translational regulation altering the total quantity of enzyme synthesized over hours or days in response to hormonal or nutritional status.

Membrane Transport Architecture and Passive Transport

• Plasma Membrane Structure:

  • Amphipathic phospholipid bilayer with hydrophilic polar head groups and a central hydrophobic core.

  • Embedded integral and peripheral proteins and cholesterol establish a selectively permeable barrier.

• Diffusion Barriers:

  • Hydrophobic molecules are restricted by outer leaflet polar head groups.

  • Hydrophilic molecules interact with polar heads but are blocked by the hydrophobic core.

• Osmosis:

  • Water moves across semi-permeable membranes down its concentration gradient via specialized channel proteins called aquaporins.

  • Effects on Red Blood Cells:

  • Isotonic solution: No net movement; cell volume remains stable.

  • Hypotonic solution: Water influx; cell volume increases, leading to lysis.

  • Hypertonic solution: Water efflux; cell volume decreases (crenation).

• Facilitated Diffusion (Passive Transport):

  • Net movement occurs down a concentration gradient ($\text{high} \rightarrow \text{low}$) without direct energy expenditure.

  • Employs ion channels (hydrophilic amino acid residue channels) or transporter proteins displaying saturable Michaelis-Menten kinetics ($V_{\max}$, $K_m$).

Active Transport Mechanisms

• Moves solutes against electrochemical gradients ($\text{low} \rightarrow \text{high}$) and requires energy.

• Primary Active Transport:

  • Direct hydrolysis of ATP powers molecule transport.

  • Example: $\text{Na}^+/\text{K}^+\text{-ATPase}$ pump. Hydrolyzes $1\,\text{ATP}$ to export $3\,\text{Na}^+$ out of the cell and import $2\,\text{K}^+$ into the cell against concentration gradients.

• Secondary Active Transport:

  • Indirectly dependent on ATP hydrolysis.

  • Uses potential energy stored in electrochemical ion gradients generated by primary active transporters.

  • Transporters lack intrinsic ATPase activity.

  • Symporters: Both solutes move in the SAME direction across the membrane (one with its gradient, one against).

• Antiporters: Solutes move in OPPOSITE directions across the membrane.

Glucose Transporters (GLUTs and SGLT)

• Glucose Uniporters (GLUT Family):

  • Facilitated diffusion down a concentration gradient.

  • Tissue Distribution and Kinetic Properties:

  • GLUT1: Most tissues; $K_m \approx 1\,mM$; basal uptake; insulin-insensitive.

  • GLUT2: Liver, kidneys, pancreatic $\beta$-cells; $K_m \approx 15-20\,mM$ (low affinity/high capacity); removes excess blood glucose; insulin-insensitive.

  • GLUT3: Most tissues (especially brain/neurons); $K_m \approx 1\,mM$; basal uptake; insulin-insensitive.

  • GLUT4: Skeletal muscle and adipose tissue; $K_m \approx 5\,mM$; insulin-sensitive.

  • GLUT5: Small intestine and testes; primary transporter of fructose.

• Insulin-Sensitive GLUT4 Regulation:

  • In resting muscle and fat cells, GLUT4 is stored in intracellular vesicles.

  • Insulin binding to its receptor triggers vesicle exocytosis and fusion with the plasma membrane, exposing GLUT4 for glucose uniport.

  • When insulin levels fall, GLUT4 endocytoses back into intracellular storage pools.

• Sodium-Glucose Cotransporter (SGLT):

  • Secondary active symport operating on the apical membrane of intestinal epithelial cells and renal proximal tubules.

  • Glucose is imported AGAINST its concentration gradient coupled to $2\,\text{Na}^+$ ions moving DOWN their electrochemical gradient.

  • The $\text{Na}^+$ gradient is maintained by basolateral $\text{Na}^+/\text{K}^+\text{-ATPase}$. Glucose exits into blood via GLUT2 uniporters on the basolateral membrane.

Pharmacological Drug Transport

• Orally administered drugs must dissolve in gastrointestinal fluid and cross mucosal epithelial cells to enter systemic circulation.

• Drugs targeting the central nervous system must penetrate endothelial tight junctions forming the blood-brain barrier.

Bioenergetics and Thermodynamics

• Gibbs Free Energy ($G$):   $\Delta G = \Delta H - T\Delta S$   where $\Delta H$ is change in enthalpy (heat content), $T$ is absolute temperature in Kelvin, and $\Delta S$ is change in entropy (randomness).

  • $\Delta G < 0$: Exergonic reaction (favorable, releases free energy, spontaneous).

  • $\Delta G > 0$: Endergonic reaction (unfavorable, requires energy input, non-spontaneous).

  • $\Delta G = 0$: Reaction at thermodynamic equilibrium.

• Endergonic reactions are driven in cells by coupling with strongly exergonic reactions (e.g., ATP hydrolysis).

Nutritional Bioenergetics

• Macronutrients: Required in large daily amounts (grams):

  • Carbohydrates $\rightarrow$ Monosaccharides

  • Proteins $\rightarrow$ Amino acids

  • Fats $\rightarrow$ Glycerol and fatty acids

• Micronutrients: Required in small amounts ($mg$ or $\mu g$); do not yield energy directly but serve as essential metabolic cofactors:

  • Water-Soluble Vitamins: B-complex vitamins (8 total) and Vitamin C. Readily absorbed and excreted; not stored (except Vitamin $B_{12}$). Deficiency is more common than toxicity.

  • Fat-Soluble Vitamins: Vitamins A, D, E, K. Absorbed with dietary fat in chylomicrons; stored in liver and adipose tissue. Toxicity is more likely.

  • Minerals: Inorganic elements ($Ca, Zn, Fe, Mg, P, K, Na$) necessary for structural, enzymatic, neurological, osmotic, and signaling functions.

• Adenosine Triphosphate (ATP):

  • Primary cellular energy currency: Adenosine + 3 phosphate groups.

  • Hydrolysis: $\text{ATP} + \text{H}_2\text{O} \rightarrow \text{ADP} + \text{P}_i$ ($\Delta G = -7.3\,kcal/mol$).

  • $\text{ADP} \rightarrow \text{AMP} + \text{P}_i$. Accumulation of AMP indicates a low cellular energy state.

Metabolism Overview: Catabolism vs. Anabolism

• Catabolism (Degradative):

  • Exergonic oxidative breakdown of complex nutrient molecules into energy-poor products ($CO_2, H_2O, NH_3$).

  • Captures energy as ATP and reduced coenzymes (NADH).

  • Three Stages: Hydrolysis of macromolecules $\rightarrow$ Conversion to Acetyl-CoA $\rightarrow$ Oxidation in TCA cycle & Oxidative Phosphorylation.

• Anabolism (Synthetic):

  • Endergonic synthesis of complex cellular macromolecules from simple precursor molecules.

  • Requires energy input (ATP hydrolysis) and reducing power (NADPH or NADH).

Redox Coenzymes

• Oxidation: Loss of electrons ($e^-$).

• Reduction: Gain of electrons ($e^-$).

• Nicotinamide Adenine Dinucleotide (NAD):

  • Derived from Niacin (Vitamin $B_3$).

  • Oxidized: $\text{NAD}^+$; Reduced: $\text{NADH}$.

  • $\text{NADP}^+ / \text{NADPH}$ contains an additional phosphate group; NADPH acts as the primary electron donor in anabolic biosynthesis.

• Flavin Adenine Dinucleotide (FAD):

  • Derived from Riboflavin (Vitamin $B_2$).

  • Oxidized: $\text{FAD}$; Reduced: $\text{FADH}_2$.

Metabolic Integration and Hormonal Control

• Highly interconnected metabolic pathways feature rate-limiting irreversible steps that serve as major regulatory bottlenecks.

• Major Metabolic Hormones:

  • Insulin: Anabolic hormone secreted by pancreatic $\beta$-cells during well-fed states (high blood glucose). Promotes nutrient uptake, glycogenesis, lipogenesis, and protein synthesis.

  • Glucagon: Catabolic hormone secreted by pancreatic $\alpha$-cells during fasting/starvation (low blood glucose). Promotes hepatic glycogenolysis, gluconeogenesis, and lipolysis.

  • Epinephrine: Catabolic catecholamine secreted by the adrenal medulla during acute stress ("fight-or-flight"). Promotes fuel mobilization in liver and muscle.

  • Cortisol: Catabolic glucocorticoid secreted by the adrenal cortex in response to chronic, long-term stress and injury.

Glycolysis

• Overview:

  • Cytosolic breakdown of 1 six-carbon glucose molecule into 2 three-carbon pyruvate molecules in all human tissues.

  • Net Output per Glucose: 2 Pyruvate, 2 Net ATP (4 produced, 2 consumed), 2 NADH.

  • Consists of 10 sequential reactions: 7 reversible steps and 3 irreversible regulatory steps.

• Three Irreversible Regulatory Steps:

  1. Step 1: Hexokinase / Glucokinase:

  • Phosphorylates glucose to Glucose 6-phosphate (G6P), trapping it inside the cytosol. Consumes 1 ATP.

  • Hexokinase: Found in most tissues; low $K_m$ (high affinity, active at basal glucose), low $V_{\max}$, feedback-inhibited by G6P.

  • Glucokinase (Hexokinase IV): Found in liver and pancreatic $\beta$-cells; high $K_m$ (low affinity, active only after high glucose intake/meals), high $V_{\max}$ (clears elevated glucose), not inhibited by G6P.

  1. Step 3: Phosphofructokinase-1 (PFK-1):

  • Converts Fructose 6-phosphate to Fructose 1,6-bisphosphate. Consumes 1 ATP.

  • Rate-limiting step of glycolysis!

  • Allosteric Regulation: Inhibited by high ATP and citrate; activated by AMP and Fructose 2,6-bisphosphate (F2,6BP).

  • Insulin increases F2,6BP levels (activates PFK-1); Glucagon decreases F2,6BP levels (inhibits PFK-1).

  1. Step 10: Pyruvate Kinase (PK):

  • Converts Phosphoenolpyruvate (PEP) to Pyruvate, generating 2 ATP.

  • Glucagon/PKA phosphorylates and inactivates hepatic PK; Insulin/PP1 dephosphorylates and activates PK.

Aerobic vs. Anaerobic Glycolytic Fates

• Aerobic Pathway: Pyruvate enters mitochondrial matrix $\rightarrow$ Pyruvate Dehydrogenase Complex $\rightarrow$ Acetyl-CoA $\rightarrow$ TCA Cycle & ETC ($\approx 30 - 32\,\text{ATP}$ per glucose).

• Anaerobic Pathway: Pyruvate is reduced to Lactate by Lactate Dehydrogenase (LDH), regenerating $\text{NAD}^+$ required to sustain glycolysis:   $\text{Pyruvate} + \text{NADH} + \text{H}^+ \xrightarrow{\text{LDH}} \text{Lactate} + \text{NAD}^+$

• Net energy yield under anaerobic conditions: 2 ATP per glucose.

• Occurs in poorly vascularized tissues (lens, cornea), cells lacking mitochondria (red blood cells), and intensely exercising skeletal muscle.

• Oral bacteria in plaque perform anaerobic glycolysis on dietary sugars, generating lactic acid that causes enamel demineralization and dental caries.

• Circulatory impairment/hypoxia forces anaerobic reliance, leading to potential clinical lactic acidosis.

Clinical Correlate: Pyruvate Kinase (PK) Deficiency

• Genetic defect causing reduced glycolytic ATP production.

• Red blood cells lack mitochondria and depend entirely on glycolytic ATP to power membrane ion pumps and preserve structural integrity.

• Severe ATP depletion leads to cell swelling, membrane distortion, and premature phagocytosis, resulting in chronic hemolytic anemia (fatigue, dyspnea, splenomegaly, neonatal jaundice).

Gluconeogenesis

• Overview:

  • De novo synthesis of glucose from non-carbohydrate precursors during prolonged fasting ($\ge 12\,\text{hours}$) or starvation.

  • Primary Site: Liver ($90\%$); Kidney cortex ($10\%$). Occurs mainly in cytosol (initial step in mitochondria).

  • Energy Cost: Consumes 4 ATP, 2 GTP, and 2 NADH per glucose molecule synthesized (equivalent to 6 high-energy phosphate bonds).

• Gluconeogenic Precursors:

  1. Glycerol: Derived from adipocyte triacylglycerol breakdown; converted to Dihydroxyacetone phosphate (DHAP).

  2. Glucogenic Amino Acids: Derived from muscle protein breakdown (all amino acids except Leucine and Lysine); enter as pyruvate or TCA cycle intermediates (e.g., oxaloacetate).

  3. Lactate: Generated by RBCs and anaerobic muscle; transported to liver via the Cori Cycle and converted to pyruvate by LDH.

• Four Irreversible Bypass Reactions:

  • Bypassing Pyruvate Kinase (Step 10):

  1. Pyruvate Carboxylase (PC) in mitochondria converts Pyruvate to Oxaloacetate (OAA). Requires Biotin, ATP, $CO_2$; allosterically activated by Acetyl-CoA.

  2. PEP Carboxykinase (PEPCK) in cytosol converts OAA to Phosphoenolpyruvate (PEP). Requires GTP. (OAA exits mitochondria via malate shuttle).

  • Bypassing PFK-1 (Step 3):

  1. Fructose 1,6-Bisphosphatase hydrolyzes Fructose 1,6-bisphosphate to Fructose 6-phosphate. Inhibited by AMP and F2,6BP; activated by ATP.

  • Bypassing Hexokinase/Glucokinase (Step 1):

  1. Glucose 6-Phosphatase in ER membrane hydrolyzes G6P to free Glucose, enabling glucose exit into systemic blood.

• Von Gierke Disease (GSD Type Ia):

  • Defect in Glucose 6-phosphatase.

  • Liver cannot export glucose from gluconeogenesis or glycogenolysis.

  • Causes severe fasting hypoglycemia, hepatomegaly, fatty liver, lactic acidosis, hyperuricemia, hyperlipidemia, and periodontitis.

Mitochondrial Pyruvate Oxidation and PDH Complex

• Mitochondrial Structure:

  • Outer membrane (contains porins).

  • Intermembrane space (proton accumulation zone).

  • Inner membrane (impermeable cristae folds containing ETC and ATP synthase).

  • Matrix (contains PDH complex, TCA cycle enzymes, mtDNA, ribosomes).

• Pyruvate Dehydrogenase (PDH) Complex:

  • Irreversible oxidative decarboxylation of pyruvate to Acetyl-CoA in matrix:     $\text{Pyruvate} + \text{CoA} + \text{NAD}^+ \rightarrow \text{Acetyl-CoA} + \text{CO}_2 + \text{NADH} + \text{H}^+$

  • Three Subenzymes and Five Coenzymes:

  • E1 (Pyruvate decarboxylase): Requires Thiamine Pyrophosphate (TPP / Vitamin $B_1$). Releases $CO_2$.

  • E2 (Dihydrolipoyl transacetylase): Requires Lipoic acid and Coenzyme A. Produces Acetyl-CoA.

  • E3 (Dihydrolipoyl dehydrogenase): Requires FAD and $\text{NAD}^+$. Generates NADH.

  • Regulation:

  • PDH Kinase: Phosphorylates and INACTIVATES E1. Activated by ATP, Acetyl-CoA, NADH; inhibited by Pyruvate.

  • PDH Phosphatase: Dephosphorylates and ACTIVATES E1. Activated by $Ca^{2+}$.

• Clinical Deficiencies: Congenital E1 deficiency leads to Congenital Lactic Acidosis and neurodegeneration. Thiamine deficiency ($B_1$) or Arsenic poisoning (binds lipoic acid) inactivates PDH.

Citric Acid Cycle (TCA Cycle)

• Regulated Steps:

  1. Citrate Synthase: Acetyl-CoA ($2C$) + Oxaloacetate ($4C$) $\rightarrow$ Citrate ($6C$). Inhibited by ATP, NADH, Succinyl-CoA.

  2. Isocitrate Dehydrogenase (Rate-limiting step!): Isocitrate $\rightarrow$ $\alpha$-Ketoglutarate + $CO_2$ + NADH. Activated by ADP, $Ca^{2+}$; inhibited by ATP, NADH.

  3. $\alpha$-Ketoglutarate Dehydrogenase Complex: $\alpha$-Ketoglutarate $\rightarrow$ Succinyl-CoA + $CO_2$ + NADH. Requires TPP, lipoic acid, FAD, $\text{NAD}^+$, CoA. Activated by $Ca^{2+}$; inhibited by Succinyl-CoA, NADH.

• Yield per Acetyl-CoA: 3 NADH, 1 $\text{FADH}_2$, 1 GTP, 2 $CO_2$.

Oxidative Phosphorylation and Electron Transport Chain

• Electron Transport Chain (ETC):

  • Inner mitochondrial membrane process transferring electrons along increasing reduction potential toward Oxygen ($O_2$), the final electron acceptor.

  • Complex I (NADH Dehydrogenase): Accepts $2e^-$ from NADH; pumps $4\text{H}^+$ into intermembrane space; transfers $2e^-$ to Coenzyme Q (CoQ).

  • Complex II (Succinate Dehydrogenase): Accepts $2e^-$ from $\text{FADH}_2$; transfers $2e^-$ to CoQ; pumps NO protons.

  • Complex III (Cytochrome $bc_1$ complex): Accepts $2e^-$ from CoQ; pumps $4\text{H}^+$; passes electrons to Cytochrome c (Cyt c).

  • Complex IV (Cytochrome c Oxidase): Accepts $2e^-$ from Cyt c; pumps $2\text{H}^+$; transfers $2e^-$ to $O_2$ ($2\text{H}^+ + \frac{1}{2} O_2 + 2e^- \rightarrow H_2O$).

• ATP Synthase ($F_0F_1$ Complex):

  • $10\text{H}^+$ pumped per NADH; $6\text{H}^+$ pumped per $\text{FADH}_2$.

  • Proton-motive force drives $\text{H}^+$ flow through $F_0$ channel, rotating the catalytic $F_1$ subunit in the matrix to synthesize ATP from ADP $+ \text{P}_i$.

  • Energy Yield: $\approx 2.5\,\text{ATP}$ per NADH; $\approx 1.5\,\text{ATP}$ per $\text{FADH}_2$. Total yield per aerobic glucose: $\approx 30 - 32\,\text{ATP}$.

• Inhibitors and Uncouplers:

  • ETC Inhibitors: Amytal & Rotenone (Complex I), Antimycin A (Complex III), Cyanide ($CN^-$) & Carbon Monoxide ($CO$) (Complex IV).

  • ATP Synthase Inhibitor: Oligomycin (closes $F_0$ proton channel).

  • Uncouplers: Dissipate proton gradient as heat without blocking ETC (e.g., UCP1/Thermogenin in brown fat, 2,4-Dinitrophenol, high-dose aspirin).

• Mitochondrial Myopathies:

  • mtDNA mutations ($10\times$ higher mutation rate than nuclear DNA) impair oxidative phosphorylation.

  • Clinical manifestations: Muscle weakness, fatigue, Ptosis (drooping eyelids), Ophthalmoplegia (inability to move eyes without turning head), dysphagia, dysarthria, periodontitis, pulpitis.

G-Protein Coupled Receptor (GPCR) Signaling

• GPCR Structure:

  • 7-transmembrane $\alpha$-helical domain receptor coupled to a heterotrimeric G-protein ($\alpha, \beta, \gamma$ subunits).

  • Inactive state: $\alpha$ subunit binds GDP. Ligand binding causes conformational change inducing GDP $\rightarrow$ GTP exchange on $G_\alpha$, causing $G_\alpha\text{-GTP}$ dissociation from $G_{\beta\gamma}$.

• Adenylyl Cyclase Pathway ($G_s / G_i$):

  • Active $G_{\alpha s}$ stimulates Adenylyl Cyclase; $G_{\alpha i}$ inhibits Adenylyl Cyclase.

  • Adenylyl Cyclase converts ATP to cyclic AMP (cAMP).

  • cAMP binds regulatory subunits of Protein Kinase A (PKA), releasing active catalytic subunits that phosphorylate Ser/Thr residues on target proteins.

  • Signal Termination: Intrinsic GTPase activity of $G_a$ hydrolyzes GTP $\rightarrow$ GDP; phosphodiesterase breaks down cAMP $\rightarrow$ AMP.

  • Bacterial Toxins:

  • Cholera Toxin: Inhibits $G_{\alpha s}$ GTPase activity $\rightarrow$ permanently active $G_s \rightarrow$ massive cAMP elevation $\rightarrow$ severe watery diarrhea.

  • Pertussis Toxin: Inactivates $G_{\alpha i} \rightarrow$ adenylyl cyclase cannot be turned off $\rightarrow$ excess cAMP $\rightarrow$ whooping cough.

• Phospholipase C (PLC) Pathway ($G_q$):

  • Active $G_{\alpha q}$ activates Phospholipase C (PLC).

  • PLC cleaves membrane $\text{PIP}_2$ into two second messengers:

  1. Inositol 1,4,5-trisphosphate ($\text{IP}_3$): Soluble messenger; opens ER $\text{Ca}^{2+}$ channels, releasing $\text{Ca}^{2+}$ into cytosol.

  2. Diacylglycerol (DAG): Membrane-bound messenger; activates Protein Kinase C (PKC) in concert with $Ca^{2+}$.

  • Free $Ca^{2+}$ also binds Calmodulin to activate downstream enzymes.

Catalytic Receptor Signaling

• Transmembrane proteins with intrinsic or associated Tyrosine Kinase activity.

• Ligand binding induces receptor dimerization and autophosphorylation on cytoplasmic Tyrosine residues.

• Phospho-Tyr residues recruit adaptor proteins containing SH2 (Src Homology 2) and SH3 domains.

• Ras / MAP Kinase Cascade:

  • Phospho-Tyr recruits Grb2 (SH2) and SOS (GEF).

  • SOS activates monomeric G-protein Ras ($Ras\text{-GDP} \rightarrow Ras\text{-GTP}$).

  • $Ras\text{-GTP}$ activates Raf (MAPKKK) $\rightarrow$ MEK (MAPKK) $\rightarrow$ MAPK (ERK) $\rightarrow$ nuclear translocation $\rightarrow$ transcription factor phosphorylation $\rightarrow$ cell proliferation.

• STAT Pathway:

  • STAT proteins dock at phospho-Tyr via SH2 domains, undergo tyrosine phosphorylation by receptor kinase, dimerize, translocate to nucleus, and stimulate transcription.

• PI3 Kinase / Akt Pathway:

  • PI3K binds phospho-Tyr and converts $\text{PIP}_2 \rightarrow \text{PIP}_3$.

  • $\text{PIP}_3$ recruits and activates Akt (Protein Kinase B), which phosphorylates Bad (inhibiting apoptosis/promoting cell survival).

  • Terminated by PTEN phosphatase.

• Insulin Receptor Signaling:

  • Pre-formed $\alpha_2\beta_2$ tetramer linked by disulfide bonds.

  • Insulin binding activates $\beta$-subunit tyrosine kinase activity $\rightarrow$ autophosphorylation $\rightarrow$ phosphorylation of Insulin Receptor Substrates (IRS).

  • IRS activates PI3K/Akt (GLUT4 translocation, glycogenesis) and Ras/MAPK (gene expression/growth).

Steroid Hormone Signaling

• Nuclear-Initiated Steroid Signaling (NISS - Classical):

  • Lipophilic steroid hormones cross plasma membrane and bind intracellular receptors in cytosol or nucleus.

  • Receptor Domains: $NH_2$-terminal regulatory, DNA-binding domain (zinc finger motif), carboxyl-terminal ligand-binding domain.

  • Ligand-receptor complex dimerizes, binds Hormone Response Elements (HREs) on DNA, and regulates gene transcription (slow onset, persistent action).

• Membrane-Initiated Steroid Signaling (MISS - Rapid):

  • Steroid receptors localized in plasma membrane caveolae.

  • Ligand binding induces association with G-proteins, Src, Ras, or PI3K, causing rapid non-genomic protein phosphorylation (seconds to minutes).

Carbohydrate Digestion and Absorption

• Classification:

  • Monosaccharides: Hexoses ($C_6H_{12}O_6$: Glucose, Galactose, Fructose).

  • Disaccharides:

  • Lactose: Glucose $+ \text{Galactose}$ ($\beta 1\rightarrow 4$ linkage).

  • Sucrose: Glucose $+ \text{Fructose}$ ($\alpha 1\rightarrow 2$ linkage).

  • Maltose: Glucose $+ \text{Glucose}$ ($\alpha 1\rightarrow 4$ linkage).

• Digestion Pathway:

  • Mouth: Salivary $\alpha$-amylase hydrolyzes internal $\alpha 1\rightarrow 4$ glycosidic bonds.

  • Stomach: Acidic pH ($1 - 3$) denatures salivary $\alpha$-amylase.

  • Small Intestine: Pancreatic bicarbonate neutralizes pH ($6 - 8$); pancreatic $\alpha$-amylase continues breakdown. Brush border disaccharidases (Isomaltase, Maltase, Lactase, Sucrase, Trehalase) yield monosaccharides.

• Absorption: Glucose and Galactose enter enterocytes via SGLT1 (secondary active transport); Fructose enters via GLUT5. All exit into portal circulation via GLUT2.

• Lactase Deficiency (Lactose Intolerance):

  • $>60\%$ of human adults experience age-dependent decline in lactase.

  • Undigested lactose draws water osmotically into colon and is fermented by gut microbiota into gases ($H_2, CO_2, CH_4$), causing abdominal cramps, flatulence, and osmotic diarrhea.

Glycogen Metabolism (Glycogenesis and Glycogenolysis)

• Glycogenesis (Synthesis):

  • Occurs in cytosol of liver and muscle.

  • Activated Substrate: UDP-Glucose ($\text{G1P} + \text{UTP} \rightarrow \text{UDP-Glucose} + \text{PP}_i$).

  • Primer: Glycogenin protein autoglucosylates at Tyrosine-194.

  • Glycogen Synthase: Key rate-limiting enzyme; forms $\alpha 1\rightarrow 4$ linear bonds at non-reducing ends.

  • Branching Enzyme (4:6 Transferase): Cleaves $6 - 8$ glucosyl units from linear chain and reattaches them via $\alpha 1\rightarrow 6$ glycosidic linkage.

• Glycogenolysis (Breakdown):

  • Glycogen Phosphorylase: Rate-limiting enzyme; phosphorolytically cleaves $\alpha 1\rightarrow 4$ bonds to yield Glucose 1-phosphate (G1P). Requires Pyridoxal Phosphate (PLP / Vitamin $B_6$).

  • Debranching Enzyme: Dual activity:

  1. Glucanotransferase: Transfers outer 3 of 4 branch residues to main chain.

  2. Amylo-$\alpha 1,6$ glucosidase: Hydrolytically cleaves remaining $\alpha 1\rightarrow 6$ linked glucose, releasing free glucose.

  • Organ Differences: Liver converts G1P $\rightarrow$ G6P $\rightarrow$ Glucose via Glucose 6-Phosphatase to maintain blood glucose. Muscle lacks Glucose 6-Phosphatase; G6P enters muscle glycolysis for ATP synthesis.

• Hormonal Regulation:

  • Insulin: Dephosphorylates enzymes $\rightarrow$ Glycogen Synthase is ACTIVE; Glycogen Phosphorylase is INACTIVE.

  • Glucagon/Epinephrine: PKA phosphorylates enzymes $\rightarrow$ Glycogen Synthase is INACTIVE; Glycogen Phosphorylase is ACTIVE.

• Glycogen Storage Diseases:

  • McArdle Disease (GSD Type V): Skeletal muscle glycogen phosphorylase deficiency (muscle cramps, myoglobinuria, exercise intolerance).

  • Hers Disease (GSD Type VI): Liver glycogen phosphorylase deficiency (mild fasting hypoglycemia, hepatomegaly).

Protein Metabolism and Nitrogen Balance

• Amino Acid Pool:

  • Free amino acid pool ($\approx 100\,g$) maintained by protein degradation ($\approx 400\,g/\text{day}$), dietary protein digestion ($\approx 100\,g/\text{day}$), and nonessential amino acid synthesis ($\approx 30\,g/\text{day}$).

  • Protein Turnover: Equal synthesis and degradation ($\approx 400\,g/\text{day}$).

• Nitrogen Balance ($N_{\text{in}} - N_{\text{out}}$):

  • Neutral Balance: Healthy adults.

  • Positive Balance: Anabolism ($N_{\text{in}} > N_{\text{out}}$; growth, pregnancy, tissue repair).

  • Negative Balance: Catabolism ($N_{\text{in}} < N_{\text{out}}$; starvation, trauma, burns, fever, infection).

• Protein Digestion and Zymogens:

  • Stomach: $HCl$ denatures proteins; activates Pepsinogen $\rightarrow$ Pepsin.

  • Pancreas/Small Intestine: Enteropeptidase activates Trypsinogen $\rightarrow$ Trypsin, which autocatalytically activates Chymotrypsinogen, Procarboxypeptidases, and Proelastase.

  • Celiac Disease: Autoimmune damage to small intestine villi triggered by dietary gluten, leading to malabsorption.

• Amino Acid Classification:

  • Essential (10): Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Leucine, Lysine.

  • Nonessential (5): Alanine, Asparagine, Aspartate, Glutamate, Serine.

  • Conditionally Essential: Cysteine, Glutamine, Glycine, Proline, Tyrosine.

Nitrogen Elimination and the Urea Cycle

• Transamination:

  • Reversible amino group transfer to $\alpha$-ketoglutarate, forming Glutamate and an $\alpha$-ketoacid. Requires Pyridoxal Phosphate (PLP / Vitamin $B_6$).

  • Alanine Transaminase (ALT) and Aspartate Transaminase (AST) serve as key clinical serum markers for hepatic injury.

• Nitrogen Transport to Liver:

  • Toxic free $NH_3$ is transported safely as Glutamine (via Glutamine Synthetase) or Alanine (via Glucose-Alanine Cycle).

• Oxidative Deamination:

  • Hepatic Glutamate Dehydrogenase releases free $NH_4^+$ and $\alpha$-ketoglutarate in mitochondrial matrix:     $\text{Glutamate} + \text{NAD(P)}^+ + H_2O \rightleftharpoons \alpha\text{-Ketoglutarate} + \text{NAD(P)H} + NH_4^+$

• Urea Cycle (Krebs-Henseleit Cycle):

  • Converts $NH_3$ into non-toxic Urea in hepatocytes (matrix + cytosol).

  • Key Reactions:

  1. Carbamoyl Phosphate Synthetase I (CPS I): Rate-limiting step in matrix:        $CO_2 + NH_3 + 2\,\text{ATP} \rightarrow \text{Carbamoyl Phosphate} + 2\,\text{ADP} + \text{P}_i$

     • Absolutely requires allosteric activator N-Acetylglutamate (NAG).

  2. Ornithine Transcarbamylase (OTC): Combines Carbamoyl Phosphate + Ornithine $\rightarrow$ Citrulline.

  3. Citrulline enters cytosol; combines with Aspartate via Argininosuccinate Synthetase $\rightarrow$ Argininosuccinate.

  4. Argininosuccinate Lyase cleaves Argininosuccinate $\rightarrow$ Arginine + Fumarate.

  5. Arginase cleaves Arginine $\rightarrow$ Urea + Ornithine.

• Hyperammonemia:

  • Normal blood $NH_3$: $5 - 35\,\mu M$. Elevated $NH_3$ ($>100\,\mu M$) is toxic to CNS, causing cerebral edema, tremors, lethargy, coma, and death.

  • Acquired: Caused by liver cirrhosis, viral hepatitis, or hepatotoxins (acetaminophen overdose).

  • Congenital: Ornithine Transcarbamylase (OTC) Deficiency (X-linked recessive). Treatment includes low-protein diet, nitrogen scavengers (Sodium Phenylbutyrate), and arginine supplementation.

Carbon Skeleton Catabolism and Inborn Errors

• Glucogenic Amino Acids: Yield glucose precursors (pyruvate, OAA, $\alpha$-KG, succinyl-CoA, fumarate).

• Ketogenic Amino Acids: Yield Acetoacetate or Acetyl-CoA (Leucine and Lysine are strictly ketogenic).

• Phenylketonuria (PKU):

  • Deficiency of Phenylalanine Hydroxylase (PAH) or tetrahydrobiopterin ($BH_4$) cofactor.

  • Phenylalanine accumulates and converts to toxic phenylketones (phenyllactate, phenylacetate, phenylpyruvate), producing a "musty" urine odor.

  • Causes severe intellectual disability and hypopigmentation.

  • Treatment: Newborn screening and dietary Phenylalanine restriction with Tyrosine supplementation.

• Maple Syrup Urine Disease (MSUD):

  • Deficiency in Branched-Chain $\alpha$-Keto Acid Dehydrogenase (BCKD) complex.

  • Impairs degradation of Branched-Chain Amino Acids (BCAAs: Leucine, Isoleucine, Valine).

  • Accumulation of BCAAs and keto-acids causes severe ketoacidosis, neurodegeneration, coma, and urine with a distinct maple syrup odor (due to isoleucine metabolites).

• Specialized Nitrogenous Molecules:

  • Nucleotides: Formed using Glutamine, Aspartate, Glycine.

  • Heme: Formed from Glycine and Succinyl-CoA.

  • Creatine: Formed from Glycine and Arginine (ATP buffer in muscle/brain).

  • Tyrosine Derivatives: Melanin (tyrosinase defect causes Albinism) and Catecholamines (Dopamine, Norepinephrine, Epinephrine).