Comprehensive Lecture Notes: Medical Biochemistry

Fundamentals of Biochemistry and Cellular Organization

• Definition of Biochemistry: Biochemistry is the science concerning the chemical basis of life (derived from the Greek bios, meaning life).

• Interdisciplinary Connections: Knowledge of biochemistry is foundational to all natural sciences and medical disciplines, including:

  • Genetics
  • Physiology
  • Immunology
  • Pharmacology
  • Toxicology
  • Pathology
  • Pharmacy

• Impact on Health and Nutrition: Biochemical research informs nutrition and preventive medicine. Maintaining optimal health requires the dietary intake of balanced amounts of chemical compounds, including vitamins, specific amino acids, essential fatty acids, mineral substances, and water.

• Cellular Organelles and Structural Layout:

  • 1 - Cytoplasm (Cytoplazma): Internal fluid environment of the cell.
  • 2 - Rough Endoplasmic Reticulum (Siateczka śródplazmatyczna szorstka): Membrane network studded with ribosomes.
  • 3 - Ribosomes (Rybosomy): Molecular machines responsible for translation.
  • 4 - Chromatin (Chromatyna): Decondensed DNA-protein complex within the nucleus.
  • 5 - Nucleolus (Jąderko): Subnuclear structure involved in ribosome biogenesis.
  • 6 - Golgi Apparatus (Aparat Golgiego): Membrane-bound organelle involved in protein sorting and post-translational modification.
  • 7 - Lysosomes (Lizosomy): Degradative organelles containing hydrolytic enzymes.
  • 8 - Plasma Membrane (Błona komórkowa): Phospholipid bilayer separating internal and external environments.
  • 9 - Mitochondria (Mitochondrium): Powerhouse organelles responsible for cellular respiration.
  • 10 - Cell Nucleus (Jądro komórkowe): Membrane-bound compartment housing the genome.

• Major Functions of Cellular Compartments:

  • Cell Nucleus: Location of chromosomes; site of DNA-dependent RNA synthesis (transcription).
  • Mitochondria: Site of the citric acid cycle (TCA cycle) and oxidative phosphorylation.
  • Ribosomes: Primary site of protein biosynthesis (translation).
  • Endoplasmic Reticulum: Membrane-bound ribosomes serve as the principal site for protein synthesis; responsible for lipid biosynthesis and oxidation of xenobiotics via the cytochrome P-450 enzyme system.
  • Lysosome: Site containing numerous hydrolytic enzymes for intracellular digestion.
  • Plasma Membrane: Regulates molecule transport into and out of cells; mediates intracellular adhesion and signal exchange.
  • Golgi Apparatus: Directs intracellular protein distribution/sorting; site of glycosylation reactions and sulfate formation reactions.
  • Peroxisome: Site for the degradation of specific fatty acids.
  • Cytoskeleton: Composed of microfilaments, microtubules, and intermediate filaments; provides structural framework and motility.
  • Cytosol: Enzymatic fluid phase containing enzymes for glycolysis and fatty acid synthesis.

Chemical Composition of the Human Body

• Elemental Composition (Percentage of Total Dry Mass):

  • Primary Organic Elements:
  • Carbon ($\text{C}$): $50\%$
  • Oxygen ($\text{O}$): $20\%$
  • Hydrogen ($\text{H}$): $10\%$
  • Nitrogen ($\text{N}$): $8.5\%$
  • Macroelements:
  • Calcium ($\text{Ca}$): $4\%$
  • Phosphorus ($\text{P}$): $2.5\%$
  • Potassium ($\text{K}$): $1\%$
  • Sulfur ($\text{S}$): $0.8\%$
  • Chlorine ($\text{Cl}$): $0.4\%$
  • Sodium ($\text{Na}$): $0.4\%$
  • Magnesium ($\text{Mg}$): $0.1\%$
  • Microelements / Trace Elements:
  • Iron ($\text{Fe}$): $0.01\%$
  • Manganese ($\text{Mn}$): $0.001\%$
  • Iodine ($\text{I}$): $0.0005\%$

• Overall Chemical Composition of a Standard $65\,\text{kg}$ Human Body:

  • Water ($\text{H}_2\text{O}$): $42\,\text{kg}$ ($60\%$ of total body mass)
  • Proteins: $12\,\text{kg}$ ($17\%$ of total body mass)
  • Lipids (Fats): $10\,\text{kg}$ ($14\%$ of total body mass)
  • Carbohydrates: $1\,\text{kg}$ ($1.5\%$ of total body mass)
  • Nucleic Acids and Other Compounds (e.g., DNA, RNA): $1\,\text{kg}$ ($1.5\%$ of total body mass)
  • Mineral Components: $4\,\text{kg}$ ($6\%$ of total body mass)

• Biomolecules and Their Building Blocks:

  • DNA: Constructed from deoxynucleotide monomers; serves as genetic material.
  • RNA: Constructed from ribonucleotide monomers; functions as a template in protein biosynthesis.
  • Proteins: Constructed from amino acid monomers; execute diverse cellular functions including catalysis (enzymes).
  • Polysaccharides (e.g., Glycogen): Constructed from glucose monomers; provide short-term energy storage.
  • Lipids: Built with fatty acid components; fulfill structural roles as cellular membrane constituents.

Water, Electrolytes, and Acid-Base Balance

• Internal Homeostasis: Maintaining the constancy of the internal environment depends on:

  • Precise spatial distribution of water.
  • Maintenance of physiological $\text{pH}$.
  • Regulation of concentrations of key electrolytes ($\text{Na}^+$, $\text{K}^+$, $\text{Ca}^{2+}$, $\text{Mg}^{2+}$).

• Body Fluid Compartments:

  • Extracellular Fluid (ECF): Represents $\frac{1}{3}$ of total body water ($25\%$ of ECF resides in blood plasma).
  • Intracellular Fluid (ICF): Represents $\frac{2}{3}$ of total body water.

• Physiological Water Regulation: Water balance is governed by the hypothalamus, antidiuretic hormone (ADH / vasopressin), and renal excretion mechanisms.

• Substance Properties in Water:

  • Hydrophilic: Possessing high affinity for water molecules.
  • Hydrophobic: Incapable of interacting with or dissolving in water.

• Water Dissociation and $\text{pH}$: Water molecules undergo slight, physiologically significant dissociation:   $\text{H}_2\text{O} \rightleftharpoons \text{H}^+ + \text{OH}^-$

  • $\text{pH}$ is defined as the negative logarithm of hydrogen ion activity/concentration:     $\text{pH} = -\log[\text{H}^+]$

• Acid-Base Balance Disorders and Compensatory Responses:

  • Respiratory Acidosis: Primary change is elevated $\text{PCO}_2$ causing reduced $\text{pH}$. Compensation involves renal reabsorption of bicarbonate ($\uparrow \text{HCO}_3^-$).
  • Metabolic Alkalosis: Primary change is elevated $\text{HCO}_3^-$ causing increased $\text{pH}$. Compensation involves hypoventilation to retain carbon dioxide ($\uparrow \text{PCO}_2$).
  • Respiratory Alkalosis: Primary change is decreased $\text{PCO}_2$ causing increased $\text{pH}$. Compensation involves renal excretion of bicarbonate ($\downarrow \text{HCO}_3^-$).
  • Metabolic Acidosis: Primary change is decreased $\text{HCO}_3^-$ causing reduced $\text{pH}$. Compensation involves hyperventilation to reduce carbon dioxide ($\downarrow \text{PCO}_2$).

Amino Acids: Structure, Classification, and Properties

• General Structure: Proteinogenic amino acids possess a central $\alpha$-carbon linked to an amino group ($-\text{NH}_2$), a carboxyl group ($-\text{COOH}$), a hydrogen atom ($-\text{H}$), and a distinctive side chain ($-\text{R}$).

• Zwitterion (Dipolar Ion): In aqueous solution at physiological $\text{pH}$, amino acids exist as zwitterions, bearing both a positive charge on the amino group ($-\text{NH}_3^+$) and a negative charge on the carboxyl group ($-\text{COO}^-$).

• Classification by Side Chain Properties:

  • Nonpolar (Hydrophobic) Side Chains:
  • Glycine (Gly / G)
  • Alanine (Ala / A)
  • Valine (Val / V)
  • Leucine (Leu / L)
  • Isoleucine (Ile / I)
  • Phenylalanine (Phe / F)
  • Tryptophan (Trp / W)
  • Methionine (Met / M)
  • Proline (Pro / P)
  • Uncharged Polar Side Chains:
  • Serine (Ser / S) — contains hydroxyl group ($-\text{CH}_2\text{OH}$)
  • Threonine (Thr / T) — contains hydroxyl group ($-\text{CH}(\text{OH})\text{CH}_3$)
  • Tyrosine (Tyr / Y) — contains phenolic hydroxyl group
  • Cysteine (Cys / C) — contains sulfhydryl group ($-\text{CH}_2\text{SH}$)
  • Asparagine (Asn / N) — contains amide group
  • Glutamine (Gln / Q) — contains amide group
  • Negatively Charged (Acidic) Polar Side Chains:
  • Aspartate / Aspartic acid (Asp / D) — contains carboxylate ($-\text{COO}^-$)
  • Glutamate / Glutamic acid (Glu / E) — contains carboxylate ($-\text{COO}^-$)
  • Positively Charged (Basic) Polar Side Chains:
  • Lysine (Lys / K) — contains primary amino group ($-\text{NH}_3^+$)
  • Arginine (Arg / R) — contains guanidino group
  • Histidine (His / H) — contains imidazole ring

• Table of the 20 Proteinogenic Amino Acids:

  • Alanine: Ala | A
  • Arginine: Arg | R
  • Asparagine: Asn | N
  • Aspartate: Asp | D
  • Cysteine: Cys | C
  • Glutamine: Gln | Q
  • Glutamate: Glu | E
  • Glycine: Gly | G
  • Histidine: His | H
  • Isoleucine: Ile | I
  • Leucine: Leu | L
  • Lysine: Lys | K
  • Methionine: Met | M
  • Phenylalanine: Phe | F
  • Proline: Pro | P
  • Serine: Ser | S
  • Threonine: Thr | T
  • Tryptophan: Trp | W
  • Tyrosine: Tyr | Y
  • Valine: Val | V

• Amphoteric Character and Titration Dynamics:

  • Amino acids act as weak acids and weak bases in aqueous solutions.
  • Acidic conditions favor protonation ($\text{H}^+$ binding) of amino groups, imidazole rings, and guanidino groups, converting the molecule into a cation.
  • Basic conditions favor proton dissociation from carboxyl groups, converting the molecule into an anion.
  • Isoelectric Point ($\text{pI}$): The specific $\text{pH}$ value at which the net electrical charge of an amino acid is zero.
  • Nonpolar amino acids: $\text{pI} \approx 6$
  • Acidic polar amino acids: $\text{pI} \approx 3$
  • Basic polar amino acids: $\text{pI} \approx 10$
  • Henderson-Hasselbalch Equation for Amino Acid Dissociation:$\text{pH} = \text{pK}_1 + \log\left(\frac{[\text{A}^-]}{[\text{HA}]}\right)$
  • For Alanine titration: $\text{pK}_1 = 2.3$ (carboxyl pair $\text{-COOH} / \text{-COO}^-$ buffering zone); $\text{pK}_2 = 9.1$ (amino pair $\text{-NH}_3^+ / \text{-NH}_2$ buffering zone). At $\text{pH} = 2.3$, equal concentrations of $\text{-COOH}$ and $\text{-COO}^-$ exist in solution.

Biologically Active Peptides

• Glutathione:

  • Tripeptide composed of Glutamate, Cysteine, and Glycine ($\text{Glu-Cys-Gly}$).
  • Features an atypical peptide bond formed between the $\gamma$-carboxyl group of glutamate and the amino group of cysteine. Glutamate retains free $\alpha$-amino and $\alpha$-carboxyl groups at the N-terminus.
  • Reduced Form (GSH): Contains a free sulfhydryl group ($-\text{SH}$).
  • Oxidized Form (GSSG): Formed by oxidation with loss of hydrogen atoms, linking two glutathione molecules via a disulfide bond ($-\text{S-S}-$).
  • Serves a vital role in cellular oxidation-reduction reactions.

• Kinins:

  • Low-molecular-weight peptides regulating vascular tone and muscle contraction:
  • Kallidin: Decapeptide sequence: $\text{Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg}$.
  • Bradykinin: Nonapeptide sequence: $\text{Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg}$.
  • Both peptides induce vasodilation of blood vessels while contracting gastrointestinal smooth muscle.

• Angiotensins:

  • Angiotensin I: Inactive decapeptide precursor: $\text{Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu}$.
  • Cleavage of the C-terminal dipeptide ($\text{His-Leu}$) produces Angiotensin II, an active octapeptide: $\text{Asp-Arg-Val-Tyr-Ile-His-Pro-Phe}$.
  • Physiological Actions of Angiotensin II:
  • Constricts smooth muscle of small blood vessels.
  • Increases myocardial contractility.
  • Regulates hormone biosynthesis and release.
  • Participates in fluid and electrolyte balance.
  • Elevates systemic arterial blood pressure.
  • Enhances sympathetic nervous system activity.

• Enkephalins:

  • Endogenous opioid pentapeptides present in the brain exhibiting potent analgesic effects:
  • Met-enkephalin: $\text{Tyr-Gly-Gly-Phe-Met}$
  • Leu-enkephalin: $\text{Tyr-Gly-Gly-Phe-Leu}$

• Endorphins:

  • Analgesic peptides synthesized in the pituitary gland, derived from $\beta$-lipotropin ($91$ amino acid precursor):
  • $\alpha$-endorphin: $16$ amino acids.
  • $\beta$-endorphin: $31$ amino acids (N-terminal sequence identical to $\alpha$-endorphin).
  • $\gamma$-endorphin: $27$ amino acids ($4$ amino acids shorter than $\beta$-endorphin).

• Oxytocin and Vasopressin:

  • Cyclic nonapeptides synthesized in the posterior pituitary gland, differing by two amino acid residues:
  • Oxytocin: Sequence: $\text{Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly}$ (contains an intramolecular disulfide bond between Cys residues). Stimulates uterine smooth muscle contractions during labor.
  • Vasopressin (Antidiuretic Hormone / ADH): Sequence: $\text{Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly}$ (contains an intramolecular disulfide bond). Enhances water reabsorption in renal tubules; at pharmacological concentrations, causes vasoconstriction and elevates blood pressure.

Proteins: Structure, Function, and Molecular Pathology

• Peptide Bond Formation: Proteins consist of amino acid monomers linked linearly by peptide bonds. Formed via a condensation reaction between the $\alpha$-carboxyl group of one amino acid and the $\alpha$-amino group of another, eliminating water:   $\text{R}_1\text{-COOH} + \text{R}_2\text{-NH}_2 \rightarrow \text{R}_1\text{-CO-NH-R}_2 + \text{H}_2\text{O}$

• Stabilizing Bonds and Levels of Protein Structure:

  • 1. Primary ($1^\circ$) Structure: Linear sequence of amino acids held together by covalent peptide bonds.
  • 2. Secondary ($2^\circ$) Structure: Local folding patterns ($\alpha$-helix and $\beta$-pleated sheet) stabilized by hydrogen bonds between peptide backbone amide ($\text{N-H}$) and carbonyl ($\text{C=O}$) groups.     
  • 3. Tertiary ($3^\circ$) Structure: Three-dimensional folding of a single polypeptide chain stabilized by:
  • Hydrophobic interactions between nonpolar side chains (e.g., Ile, Leu).
  • Hydrogen bonds between polar side chains (e.g., Ser).
  • Disulfide bonds (covalent linkage between two cysteine residues).
  • Electrostatic/ionic interactions (e.g., between Asp/Glu and Lys/Arg).
  • van der Waals forces.
  • 4. Quaternary ($4^\circ$) Structure: Association of multiple polypeptide subunits into an oligomeric functional complex.
  • Held together primarily by low-energy non-covalent interactions (hydrophobic, ionic, hydrogen bonds).
  • May be stabilized by disulfide bonds.
  • Collagen and elastin feature stable covalent inter-subunit cross-links.

• Hemoglobin Variants and Primary Structure Variations:

  • HbA (Normal Adult Hemoglobin): $\text{H}_2\text{N-Val-His-Leu-Thr-Pro-Glu-Glu-Lys}$
  • HbS (Sickle Cell Hemoglobin): $\text{H}_2\text{N-Val-His-Leu-Thr-Pro-Val-Glu-Lys}$ (Glutamate at position 6 replaced by neutral Valine).
  • HbC Hemoglobin: $\text{H}_2\text{N-Val-His-Leu-Thr-Pro-Lys-Glu-Lys}$ (Glutamate at position 6 replaced by positively charged Lysine).

• Molecular Pathology Example — Osteogenesis Imperfecta:

  • Replacement of single Glycine residues in the primary sequence of bone collagen by bulky amino acids (Arginine, Cysteine, Serine, or Alanine) disrupts triple-helix spatial folding.
  • Leads to osteogenesis imperfecta, characterized by severe reduction in bone mechanical strength and extreme bone fragility.

• Post-Translational Modifications:

  • Amino acid modifications tailor protein functions: N-terminal acetylation confers resistance against proteolytic degradation; Proline hydroxylation stabilizes collagen triple-helix fibers.

• Functional Categories of Proteins:

  • Enzymatic Catalysis: Virtually all cellular chemical reactions are catalyzed by protein enzymes.
  • Transport and Storage: Transferrin transports iron in plasma; Ferritin stores iron in the liver; Albumin transports free fatty acids in blood.
  • Coordinated Movement: Actin and myosin filaments slide past each other to generate muscle contraction.
  • Mechanical and Structural Support: Fibrous collagen provides structural elasticity and tensile strength to bone and tissues.
  • Immune Protection: Antibodies (immunoglobulins) selectively recognize foreign antigens.
  • Growth and Differentiation Control: Protein growth factors and hormones (e.g., Insulin) control metabolic flux and development.
  • Nerve Impulse Generation and Photoreception:
  • Color vision relies on three distinct opsin photoreceptors with specific peak absorption wavelengths: OPN1LW ($560\,\text{nm}$ - red), OPN1MW ($530\,\text{nm}$ - green), OPN1SW ($430\,\text{nm}$ - blue).
  • Photochemical retinal reaction in rod cells:       $\text{Rhodopsin (11-cis-retinal + opsin)} \xrightarrow{h\nu} \text{11-trans-retinal} + \text{opsin}$

Enzymology: Mechanisms, Kinetics, and Classification

• General Properties of Biological Catalysts:

  • Macro-molecules that accelerate reaction rates without altering reaction equilibrium.
  • Highly specific toward substrates.
  • Remain unconsumed during the reaction cycle; a single enzyme molecule processes many substrate molecules.
  • Carbonic Anhydrase Acceleration: Accelerates $\text{CO}_2$ hydration rate by $10^7$ times compared to the uncatalyzed reaction:     $\text{CO}_2 + \text{H}_2\text{O} \rightleftharpoons \text{HCO}_3^- + \text{H}^+$

• Enzyme Activity Quantification:

  • International Unit ($U$ / u): Amount of enzyme activity that converts $1\,\mu\text{mol}$ of substrate to product per minute at $30^\circ\text{C}$ under optimal $\text{pH}$ and zero-order conditions.
  • Molar Activity: Number of enzyme units ($U$) per $1\,\text{mol}$ of pure enzyme protein.

• Mechanism of Enzymatic Action and Energy Profiles:

  • Catalytic sequence:     $\text{S} + \text{E} \rightleftharpoons \text{ES} \rightleftharpoons \text{EP} \rightleftharpoons \text{P} + \text{E}$
  • Activation Energy ($\Delta G^*$): Difference between the free energy of the transition state and the free energy of the substrate. Enzymes lower $\Delta G^*$, significantly increasing reaction rate ($v$).

• Substrate Binding Models:

  • Lock and Key Model: Rigid complementation between active site geometry and substrate shape.
  • Induced Fit Model: Dynamic conformational alteration of the enzyme's active site upon substrate interaction to optimize catalytic orientation.

• Factors Influencing Enzymatic Rates:

  • Temperature: Reaction velocity increases with temperature up to an optimum ($\sim 37^\circ\text{C}-40^\circ\text{C}$), above which thermal denaturation occurs.
  • $\text{pH}$ Sensitivity: Enzymes exhibit specific $\text{pH}$ optima (e.g., Pepsin $\text{pH} \approx 2$; Amylase $\text{pH} \approx 7$; Trypsin $\text{pH} \approx 8$).

• Michaelis-Menten Kinetics:

  • Maximum Velocity ($v_{max}$): Reaction velocity reached when all active sites are fully saturated with substrate.
  • Michaelis Constant ($K_m$): Substrate concentration ($\text{mol/L}$) at which reaction velocity reaches half of $\text{v}_{max}$ ($\frac{v_{max}}{2}$).
  • Low $K_m$ indicates high enzyme-substrate affinity.
  • High $K_m$ indicates low enzyme-substrate affinity.
  • Lineweaver-Burk Double Reciprocal Plot: Plots $\frac{1}{v}$ against $\frac{1}{[S]}$.
  • Y-intercept equals $\frac{1}{v_{max}}$.
  • X-intercept equals $-\frac{1}{K_m}$.

• Enzyme Classification (6 Major Classes):

  • Class 1 — Oxidoreductases: Catalyze oxidation-reduction reactions:     $\text{A}_{red} + \text{B}_{ox} \rightleftharpoons \text{A}_{ox} + \text{B}_{red}$
  • Class 2 — Transferases: Catalyze functional group transfer between molecules:     $\text{A-B} + \text{C} \rightleftharpoons \text{A} + \text{B-C}$
  • Class 3 — Hydrolases: Catalyze cleavage of bonds with the addition of water:     $\text{A-B} + \text{H}_2\text{O} \rightleftharpoons \text{A-H} + \text{B-OH}$
  • Class 4 — Lyases: Catalyze non-hydrolytic group removal or addition without water:     $\text{A(HX)-B} \rightleftharpoons \text{A-X} + \text{BH}$
  • Class 5 — Isomerases: Catalyze intramolecular rearrangement reactions:     $\text{A} \rightleftharpoons \text{IsoA}$
  • Class 6 — Ligases (Synthetases): Catalyze bond formation coupled with ATP cleavage:     $\text{A} + \text{B} + \text{ATP} \rightleftharpoons \text{A-B} + \text{ADP} + \text{P}_i$

Coenzymes, Cofactors, and Inhibition

• Terminology:

  • Cofactor: Non-protein components required for activity, including coenzymes and inorganic metal ions ($\text{Ca}^{2+}$, $\text{Mg}^{2+}$, $\text{Zn}^{2+}$, $\text{Fe}^{2+/3+}$).
  • Apoenzyme: Catalytically inactive protein component lacking its cofactor.
  • Holoenzyme: Catalytically active complex consisting of apoenzyme plus bound cofactor.

• Major Coenzymes and Functions:

  • $\text{NAD}^+$ / $\text{NADP}^+$ (Nicotinamide Adenine Dinucleotide / Phosphate): Derived from Niacin (Vitamin $\text{B}_3$). Function as electron/proton acceptors for dehydrogenases.     
  • FMN / FAD (Flavin Mononucleotide / Flavin Adenine Dinucleotide): Derived from Riboflavin (Vitamin $\text{B}_2$). FMN serves as an electron/proton transporter in the respiratory chain; FAD acts as a direct hydrogen acceptor.     
  • Coenzyme A (CoA-SH): Composed of ADP, pantothenic acid (Vitamin $\text{B}_5$), and cysteamine. The cysteamine sulfhydryl group ($-\text{SH}$) forms high-energy thioester bonds ($\text{acyl}\sim\text{SCoA}$, e.g., $\text{acetyl}\sim\text{SCoA}$), activating acyl groups for ester biosynthesis (acetylcholine, acylglycerols) or catabolic pathways.
  • Biotin: Binds $\text{CO}_2$ to form carboxybiotin, acting as a cosubstrate in carboxylation reactions.
  • Lipoic Acid (Thioctic Acid): Eight-carbon fatty acid. Exists in reduced form (dihydrolipoic acid, free $-\text{SH}$ at C6 and C8) and oxidized form (dehydrolipoic acid, intramolecular disulfide bond). Acts as an acyl/acetyl group carrier in oxidative decarboxylation of $\alpha$-keto acids.
  • Thiamine Pyrophosphate (TPP): Derived from Thiamine (Vitamin $\text{B}_1$). Participates in oxidative decarboxylation of $\alpha$-keto acids (pyruvate, $\alpha$-ketoglutarate) by binding carbonyl groups and releasing $\text{CO}_2$
  • Pyridoxal Phosphate (PLP): Derived from Pyridoxine (Vitamin $\text{B}_6$). Functions as an amino group carrier in transamination reactions.
  • Tetrahydrofolate (THF): Derived from Folic Acid. Transports one-carbon units in amino acid metabolism, purine synthesis, and transmethylation reactions.
  • Heme: Porphyrin ring system containing $\text{Fe}^{2+}$ or $\text{Fe}^{3+}$. Serves as coenzyme in cytochromes and prosthetic group in catalase, peroxidase, hemoglobin, and myoglobin.
  • Coenzyme $\text{B}_{12}$: Derived from Cobalamin (Vitamin $\text{B}_{12}$). Participates in odd-chain fatty acid metabolism and transmethylation.

• Enzyme Inhibition Mechanics:

  • Irreversible Inhibition: Inhibitor binds covalently or extremely tightly to active site residue; cannot be detached by dialysis or dilution. Example: Acetylsalicylic acid (aspirin) irreversibly inactivates cyclooxygenase, blocking prostaglandin synthesis.
  • Reversible Inhibition: Inhibitor binds non-covalently and can disassociate from the enzyme:
  • Competitive Inhibition: Inhibitor structurally resembles substrate and competes for active site binding.
    • Effects: $v_{max}$ remains unchanged; $K_m$ increases ($K_{m,\text{inh}} > K_m$).
    • Inhibition can be overcome by increasing substrate concentration ($[S]$).
    • Example: Succinate dehydrogenase is competitively inhibited by malonate, oxalate, and oxaloacetate.       
  • Non-Competitive Inhibition: Inhibitor binds allosterically at a distinct site, regardless of substrate presence.
    • Effects: $v_{max}$ decreases ($v_{max,\text{inh}} < v_{max}$); $K_m$ remains unchanged.
    • Cannot be overcome by increasing $[S]$.
  • Uncompetitive and Mixed Inhibition.

Clinical Enzymology and Isoenzymes

• Diagnostic Plasma Enzymes and Clinical Applications:

  • Aspartate Aminotransferase (AspAT): Diagnostic marker for myocardial infarction.
  • Alanine Aminotransferase (AlAT): Diagnostic marker for viral hepatitis.
  • Amylase: Diagnostic marker for acute pancreatitis.
  • Creatine Kinase (CK): Diagnostic marker for muscle diseases and myocardial infarction.
  • $\gamma$-Glutamyl Transpeptidase (GGTP): Diagnostic marker for various liver disorders.
  • Lactate Dehydrogenase (LDH Isoenzymes): Diagnostic marker for myocardial infarction and hepatic injury.
  • Lipase: Diagnostic marker for acute pancreatitis.
  • Acid Phosphatase: Diagnostic marker for metastatic prostate carcinoma.
  • Alkaline Phosphatase: Diagnostic marker for bone diseases and obstructive biliary tract liver disorders.

• Enzyme Dynamics Post-Myocardial Infarction:

  • CK-MB: Rises sharply within hours, peaking around $24\,\text{hours}$.
  • AspAT: Rises moderately, peaking at $24-48\,\text{hours}$.
  • LDH: Rises more slowly, peaking at $2-3\,\text{days}$ and staying elevated for up to $7-10\,\text{days}$.
  • Troponins: Rise rapidly and remain elevated for extended periods.

• Enzyme Substrate Specificity:

  • Absolute Specificity: Enzyme acts on only one specific substrate or stereoisomer (e.g., LDH oxidizes L-lactate to pyruvate, but does not act on D-lactate).
  • Relative Specificity: Enzyme acts on a group of structurally related compounds (e.g., Phosphatase cleaves general phosphate esters).

• Isoenzymes: Genetically distinct forms of an enzyme catalyzing the same reaction within an organism, differing in amino acid sequence, subunit composition, and kinetic parameters.

  • Lactate Dehydrogenase Isoenzymes (LDH): Tetramer formed from H and M subunits:
  • $\text{LDH}_1$ ($\text{H}_4$): Present in cardiac muscle and brain; $K_m = 1.4 \times 10^{-4}\,\text{M}$; sensitive to pyruvate inhibition.
  • $\text{LDH}_5$ ($\text{M}_4$): Present in skeletal muscle; $K_m = 10^{-3}\,\text{M}$.
  • Creatine Kinase Isoenzymes (CK): Dimer formed from B and M subunits:
  • $\text{CK-1}$ ($\text{BB}$): Present in brain tissue.
  • $\text{CK-2}$ ($\text{MB}$): Present in cardiac tissue.
  • $\text{CK-3}$ ($\text{MM}$): Present in skeletal muscle tissue.

• Pharmacological Enzyme Activators: Glucokinase activator drugs increase glucokinase affinity and $v_{max}$, increasing pancreatic insulin release and boosting hepatic glucose uptake.

Oxygen Transport Proteins: Hemoglobin and Myoglobin

• Oxygen Transport Demands: The human body consumes $\sim 500\,\text{g}$ of oxygen per day. Due to low oxygen solubility in aqueous fluids, transport requires specialized hemeproteins.

• Comparison Between Hemoglobin and Myoglobin:

  • Localization: Red blood cells vs. Skeletal/cardiac muscle tissue.
  • Function: Systemic $\text{O}_2$ transport (lungs to tissues) and $\text{CO}_2$ transport (tissues to lungs) vs. Local $\text{O}_2$ storage and intracellular transport facilitation.
  • Structure: Heterotetramer ($\alpha_2\beta_2$ in HbA; $\alpha_2\gamma_2$ in fetal HbF) vs. Monomer (1 chain).
  • Oxygen Capacity: Binds $4\,\text{O}_2$ molecules vs. Binds $1\,\text{O}_2$ molecule.
  • Ligands Transported: $\text{O}_2$, $\text{CO}_2$, $\text{H}^+$ vs. Only $\text{O}_2$
  • Affinity and Allostery: Lower affinity, pH-dependent, cooperative, allosteric vs. Higher affinity, pH-independent, non-cooperative, non-allosteric.
  • HbF vs. HbA Structural Difference: The $\gamma$-subunit of HbF contains Serine at position 21 (Ser21) instead of Histidine (His21) found in the $\beta$-subunit of HbA. This eliminates key ionic bonds, resulting in weaker BPG binding and higher $\text{O}_2$ affinity for HbF.

• Oxygen Dissociation Curves:

  • Myoglobin: Hyperbolic curve with $p_{50} = 1\,\text{mmHg}$, indicating high affinity and efficient binding at low oxygen pressures.
  • Hemoglobin: Sigmoidal curve with $p_{50} = 26\,\text{mmHg}$, indicating cooperative allosteric binding.

• Cooperativity Mechanism:

  • T (Tense) State: Deoxyhemoglobin spatial conformation; compact structure stabilized by ionic bonds with lower $\text{O}_2$ affinity.
  • R (Relaxed) State: Oxyhemoglobin spatial conformation; relaxed structure with higher $\text{O}_2$ affinity.
  • Oxygen binding sequentially breaks ionic bonds, converting T-state to R-state. Oxygen acts as a positive allosteric effector ($K_1 < K_2 < K_3 < K_4$).

• Methemoglobin: Oxidation of iron from ferrous ($\text{Fe}^{2+}$) to ferric ($\text{Fe}^{3+}$) form converts hemoglobin into methemoglobin, which is incapable of binding and transporting oxygen.

• 2,3-Bisphosphoglycerate (2,3-BPG) Regulation:

  • Byproduct of glycolysis that binds non-covalently in a 1:1 ratio to deoxyhemoglobin at an allosteric site distant from heme.
  • Acts as a negative allosteric effector, stabilizing the T-state and promoting oxygen release in peripheral tissues.
  • Intra-erythrocytic BPG levels rise during hypoxia (respiratory failure, circulatory insufficiency, anemia), facilitating oxygen unloading in tissue capillaries.

• Carbon Monoxide (CO) Toxicity:

  • CO binds $\text{Fe}^{2+}$ in heme with an affinity $\sim 200$ times greater than oxygen.
  • Competitive inhibitor of $\text{O}_2$ binding to hemoglobin and myoglobin; also inhibits cytochrome oxidase in the respiratory chain.
  • Binding CO to one subunit locks the remaining subunits in the high-affinity R-state, preventing $\text{O}_2$ unloading in peripheral tissues.
  • Tobacco smokers carry $4-8\%$ carboxyhemoglobin (HbCO). Clinical toxicity appears at $30-50\%$ HbCO saturation; $80\%$ saturation is fatal. Reversible by hyperbaric oxygen administration.

• The Bohr Effect:

  • Explains the reciprocal coupling between $\text{O}_2$, $\text{CO}_2$, and $\text{H}^+$ binding.
  • In Tissues: High $\text{CO}_2$ and $\text{H}^+$ lower $\text{pH}$, driving protonation of Hb and stabilizing the T-state, forcing $\text{O}_2$ release:     $\text{CO}_2 + \text{H}_2\text{O} \xrightleftharpoons{\text{Carbonic Anhydrase}} \text{H}_2\text{CO}_3 \rightleftharpoons \text{HCO}_3^- + \text{H}^+$
  • In Lungs: High $\text{O}_2$ partial pressure forces $\text{O}_2$ binding to Hb, releasing $\text{H}^+$, which combines with $\text{HCO}_3^-$ to yield $\text{CO}_2$ for exhalation.

Heme Metabolism: Biosynthesis, Degradation, and Clinical Conditions

• Chemical Structure of Heme:

  • Pyrrole pigment containing an $\text{Fe}^{2+}$ or $\text{Fe}^{3+}$ ion covalently linked to two nitrogen atoms of pyrrole rings.
  • Forms four coordination bonds: two with remaining ring nitrogens, one with proximal histidine in globin, and one with $\text{H}_2\text{O}$ or $\text{O}_2$
  • Nomenclature: $\text{Fe}^{2+}\text{-1,3,5,8-tetramethyl-2,4-divinyl-6,7-dipropionate-protoporphyrin IX}$.

• Pathway of Heme Biosynthesis:

  • Step 1: Condensation of Succinyl-CoA and Glycine catalyzed by $\delta$-aminolevulinic acid synthase to yield $\alpha$-amino-$\beta$-ketoadipate, which decarboxylates to $\delta$-aminolevulinic acid (ALA).     
  • Step 2: Condensation of two ALA molecules catalyzed by $\delta$-aminolevulinic acid dehydratase, releasing $2\,\text{H}_2\text{O}$ to form Porphobilinogen.     
  • Step 3: Assembly of four porphobilinogen molecules by uroporphyrinogen synthase releases $4\,\text{NH}_3$ to generate Uroporphyrinogen III.
  • Step 4: Sequential decarboxylations ($-4\text{CO}_2$, $-2\text{CO}_2$), oxidations ($-4\text{H}$, $-6\text{H}$), and insertion of $\text{Fe}^{2+}$ by Ferrochelatase yields Heme.     

• Porphyrias: Inherited or acquired enzymatic defects in heme biosynthesis leading to accumulation and urinary excretion of intermediates ($\delta$-aminolevulinic acid and porphobilinogen).

• Heme Catabolism and Bilirubin Metabolism:

  • Step 1: Senescent red blood cells (erythrocyte lifespan $\sim 120$ days) are phagocytosed by macrophages of the reticuloendothelial system (spleen, liver, tissue macrophages).
  • Step 2: Heme oxygenase cleaves heme into Biliverdin (green pigment), releasing $\text{Fe}^{3+}$ and $\text{CO}$. Biliverdin reductase reduces biliverdin to Bilirubin (orange-red pigment, poorly soluble in water).
  • Step 3: Unconjugated (indirect) bilirubin is released into blood and transported bound to albumin as a bilirubin-albumin complex.
  • Step 4: Liver parenchymal cells absorb bilirubin. The enzyme Bilirubin glucuronyltransferase conjugates bilirubin with two molecules of UDP-glucuronic acid, forming water-soluble Bilirubin diglucuronide (conjugated / direct bilirubin).
  • Step 5: Conjugated bilirubin is secreted into bile and drains into the small intestine.
  • Step 6: Intestinal bacteria deconjugate glucuronic acid and reduce bilirubin into Urobilinogen.
  • Step 7: Fates of Urobilinogen:
  • Reabsorbed in intestine to enter portal circulation (enterohepatic urobilinogen cycle).
  • Excreted via kidneys into urine after oxidation to yellow Urobilin (gives urine its normal color).
  • Oxidized by gut bacteria into brown Stercobilin and excreted in feces.

• Neonatal Jaundice: Caused by transient developmental deficiency of glucuronyltransferase activity in newborns. Enzyme activity reaches physiological adult levels after $\sim 2$ weeks. Neonatal hyperbilirubinemia is treated using phototherapy.