	Body Fluid Volume (L)	Body Fluid (%)	Body Weight (%)
Total Body Water	42	100	60
Intracellular Fluid	28	67	40
Extracellular Fluid	14	33	20
Plasma	2.8	6.6	4
Interstitial Fluid	11.2	26.4	16
Note	Extracellular Fluid = Plasma + Interstitial Fluid

Table 1. Body fluid distribution

③ Water loss: Adults need approximately 3 liters of water daily (1.5 liters from food and 1.5 liters from beverages).

Water Loss (%)	Symptoms
1 ~ 2	Thirst
3 ~ 4	Decreased physical performance, reduced urine output, fatigue
5 ~ 6	Breathing and pulse irregularities, mental confusion
8	Dizziness, severe fatigue
10 ~ 11	Heatstroke, risk of death

Table 2. Symptoms of water loss

⑶ Carbohydrate

① Cell’s main energy source

○ 4 kcal/g. Energy is stored in chemical bonds.

② Absorption rate: Monosaccharides > Disaccharides > Polysaccharides

③ Starch: Glucose polymers of plants.

④ Glycogen: Glucose Polymers of animals.

○ Store carbohydrates in glycogen form in liver and muscle.

○ Energy source exhausted in one day.

⑤ Dietary fiber (e.g., cellulose)

○ Cleanses digestive waste in the large intestine: Dietary fiber is not digested.

○ Digestive waste: Includes bacteria, toxic substances, parasites, etc.

○ Increases cholesterol absorption in the small intestine and lowers cancer risk.

○ The most abundant carbohydrate on Earth.

⑥ Detection reagent

○ Starch: Iodine-Potassium Iodide solution, iodine reaction, purple color

○ Glucose, Fructose, Maltose, etc.: Benedict’s test (yellow-red color, requires heating)

⑷ Protein

① Primary energy source and major component of cells

○ 4 kcal/g. Energy is stored in chemical bonds (the last resort energy source).

② Polymer of 20 amino acids. Amino acids are linked by peptide bonds.

③ Essential amino acids

○ Amino acids to be taken as food. 8 for adults and 10 for children.

○ Valine, Leucine, Isoleucine, Methionine, Threonine, Lysine, Phenylalanine, Tryptophan, Histidine (for child), Arginine (for child)

○ Animal Protein: Contains all essential amino acids.

○ Plant Protein: Rarely contains all essential amino acids.

○ Amino acids are hydrophilic and cannot be stored; therefore, they must be supplied daily.

④ Complete protein

○ Proteins that contain all essential amino acids: Meat, eggs

○ Plant protein: Not a complete protein; a variety of ingredients must be combined when following a vegetarian diet.

⑤ Limiting Protein

○ Proteins that lack even one essential amino acid.

⑥ Detection reagent: Biuret Test (purple color)

⑸ Fat

① Primary Energy Storage Molecule, protects vital organs, provides insulation, and serves as a reserve during famine.

○ 9 kcal/g: Energy is stored in chemical bonds.

○ Excess carbohydrates are stored as fat.

② Glycerol + Fatty acid (hydrocarbon) tail

③ Fatty Acids

○ Short-chain fatty acids (SCT): 6 or fewer carbon atoms

○ Medium-chain fatty acids (MCT): 8 to 10 carbon atoms

○ Long-chain fatty acids (LCT): 12 or more carbon atoms

○ Fatty acids are synthesized from Acetyl-CoA (C2), so they have an even number of carbon atoms.

④ Essential fatty acids: Unsaturated fatty acids that cannot be synthesized in the body.

○ Linoleic Acid and Linolenic Acid: Polyunsaturated fatty acids found in corn and safflower seed oils; involved in the production of phospholipids present in cell membranes.

○ Arachidonic Acid: A precursor to important signaling molecules such as prostaglandins.

○ Omega-3 and Omega-6: Unsaturated fatty acids found in cold-water fish (e.g., salmon, sardines) and flaxseed oil, known for their heart-protective functions.

⑤ Trans fats (Transitional fats)

○ Cis-unsaturated fatty acids can be hydrogenated to create saturated fatty acids, during which trans-unsaturated fatty acids or trans fats are formed.

○ Examples: Shortening, margarine

○ Health Impact: Increases the risk of heart disease and diabetes.

○ WHO recommendation: Trans fats should make up less than 1% of total caloric intake.

⑥ Detection reagent: Sudan III (scarlet color)

⑹ Vitamin

① Function: Generally acts as a coenzyme required for enzyme activity, regulating metabolism and physiological functions.

② Provitamin: A precursor of a vitamin that has not yet become active.

○ Example: Beta Carotene, Ergocalciferol

③ Water soluble vitamins (B, C)

○ Overview

○ Loss during cooking. Should be consumed as fresh vegetables; not stored in the body, making deficiency possible.

○ Form in food: Available as compressed tablets or packaged supplements.

○ Distribution: Dissolves and is distributed in bodily fluids such as blood and tissue fluid.

○ Excretion: Easily excreted in urine, increasing the likelihood of deficiency.

○ Riboflavin (vitamin B₂): FAD, FMN

○ Niacin (vitamin B₃): NAD⁺, NADP

○ Biotin (vitamin B₇): Present in coenzyme.

○ Folic acid (vitamin B₉): Also known as folate.

○ Function: Essential for purine nucleotide biosynthesis, a precursor for red blood cells, and an amino acid source required for cell division.

○ Folate absorbed from food enters the folate metabolism pathway and is reduced to tetrahydrofolate (THF).

○ Tetrahydrofolate provides single atoms during purine biosynthesis.

○ Methotrexate: An anti-folate agent that inhibits dihydrofolate reductase (DHFR).

○ Cobalamin (Vitamin B₁₂)

○ Nucleic acid synthesis, a component for red blood cell production.

○ Deficiency: Pernicious anemia

○ Functions with Co ions.

○ Converts homocysteine to cysteine.

○ Found in animal-based proteins.

○ Vitamin C

○ Function 1. Collagen synthesis

○ 1^st. Vitamin C acts as a coenzyme for prolyl hydroxylase.

○ 2^nd. Prolyl hydroxylase converts proline, abundant in collagen, into hydroxyproline.

○ 3^rd. Hydroxyproline forms strong bonds, enabling various binding functions in the extracellular matrix (ECM).

○ Function 2. Increases intestinal iron absorption

○ In the stomach, Fe³⁺ is reduced to Fe²⁺, and when combined with Vitamin C, iron absorption is facilitated.

○ Deficiency: Scurvy

④ Fat-soluble vitamins (A, D, E, K)

○ Overview

○ Fat-soluble vitamins are not easily excreted, which can lead to toxicity.

○ Most fat-soluble vitamins, including vitamins A and D, are stored in the liver.

○ Form in food: Packaged as oil-based gel capsules in supplements.

○ Distributed in structures like cell membrane tissues.

○ Not excreted in urine but eliminated through bile.

○ Vitamin A

○ First discovered in 1912 when Dr. Hopkins identified a growth factor in milk essential for animals.

○ A component of rhodopsin, a pigment protein found in rod cells of the retina.

○ Type 1. Retinol: Found in animal-based foods such as liver, milk, whole milk powder, butter, egg yolk, cod, liver oil, etc.

○ Type 2. Carotenoids: Orange pigments found in plant-based foods.

○ Converted into Vitamin A in the body.

○ Among carotenoids, beta-carotene has the highest activity. It is abundant in green vegetables like carrots, spinach, and seaweed.

○ Deficiency 1. Night Blindness: A condition where vision becomes impaired in dark settings after exposure to bright light.

○ Deficiency 2. Xerophthalmia: Dryness of the eyes.

○ Vitamin D (Calciferol): The only vitamin that can be synthesized by the body.

○ 1^st. Skin: 7-dehydroxycholesterol is converted to cholecalciferol (vitamin D3) upon exposure to ultraviolet rays.

○ 2^nd. Liver: Cholecalciferol is converted to 25-OH vitamin D3 by 25-hydroxylase.

○ 3^rd. Kidney: 25-OH vitamin D3 is further converted to calcitriol (1,25-(OH)2 vitamin D3) by 1α-hydroxylase.

○ 4^th. Small Intestine: Calcitriol, as the active form of vitamin D, activates calcium pumps to enhance calcium absorption.

○ Deficiency: Rickets

○ Vitamin E: Includes alpha-tocopherol and tocotrienol. Acts as a fat-soluble antioxidant and helps prevent infertility.

○ Deficiency: Aging.

○ Vitamin K: Activates blood clotting proteins.

○ Carboxylates glutamic acid residues in prothrombin during the blood clotting process.

○ Deficiency: Delayed blood clotting

⑤ Types of vitamin

Table 3. Types of vitamin

⑺ Minerals: Also known as minerals or inorganic substances.

① Overview

○ Definition: Components of living organisms that exclude the three main elements—carbon, hydrogen, and oxygen.

○ Composition: Account for approximately 4% of the human body’s composition.

○ Function: Involved in physical growth, body maintenance, and reproduction.

② Primary Functions of Inorganic Ions

○ Sodium (Na)

○ Maintains osmotic pressure in extracellular fluid.

○ Triggers action potentials.

○ Recommended intake: 7 g.

○ Excessive Intake:

○ Hypertension: Because sodium constricts blood vessels and alters hormones.

○ Increased Osmotic Pressure: Causes water to leave cells, raising intracellular acidity and disrupting protein compositions.

○ Excess Salt Consumption: Irritates the digestive tract, interfering with nutrient absorption.

○ Magnesium (Mg)

○ Importance: Mg²⁺ plays a critical role in molecular biology and polymerases.

○ ATPase

○ DNA Polymerase

○ RNA Polymerase: Requires two Mg²⁺ ions. One binds to the oxygen of the preceding 3’-OH group, and the other binds to the dNTP.

○ Ribozymes: Require Mg²⁺ and protein assistance to function.

○ Phosphorus (P)

○ A component of phospholipids and nucleic acids.

○ Phosphates act as a buffer for hydrogen ions along with carbonates.

○ Most phosphorus exists in bones and teeth in the form of calcium phosphate.

○ Distribution: Widely present in all natural foods, particularly abundant in animal-based foods such as milk, dairy products, and meat.

○ Brown rice also contains a high amount of phosphorus, but most of it exists in the form of phytic acid, which is a substance that inhibits phosphorus absorption.

○ Excess Intake: Excessive phosphorus consumption can hinder calcium absorption.

○ Chlorine (Cl)

○ Involved in regulating osmotic pressure as a counter ion to inorganic cations.

○ Present as hydrochloric acid (HCl), which maintains the acidity of gastric juice, prevents bacterial fermentation, and aids digestion.

○ Excess Intake: Can cause hypertension.

○ Potassium (K)

○ Key osmotic pressure factor in intracellular fluid.

○ Acid-Base Balance: Hypokalemia reduces H⁺ levels.

○ Essential for urea production in urine formation.

○ Excess potassium intake is excreted in urine, as the kidneys cannot store potassium as effectively as sodium.

○ Electrophysiology: Involved in membrane repolarization and establishing the resting potential.

○ Metabolism: Participates in glycogen and protein synthesis.

○ Excess Intake: Can lead to muscle weakness, paralysis, and cardiac arrest.

○ Calcium (Ca)

○ Exocytosis: Related to neurotransmitter release in nerve cells.

○ Acts as a cofactor in the blood coagulation process.

○ Involved in skeletal and cardiac muscle contraction.

○ Stabilizes cadherins in desmosomes for intercellular connections.

○ 99% of calcium is distributed in bones and teeth.

○ Second Messenger: Functions as a secondary signaling molecule.

○ When calcium ions are low: Sodium channels remain widely open, causing overactivity.

○ When calcium ions are high: Sodium channels are mostly closed, leading to reduced strength.

○ Middle-aged women with high calcium levels may experience reduced physical strength.

○ Deficiency: Osteoporosis, stunted growth

○ Excess: Kidney stones

○ Manganese (Mn)

○ Iron (Fe)

○ Hematopoiesis: Over half of the body’s iron is a component of hemoglobin.

○ Oxygen Transport: Inorganic iron salts enable rapid oxygen absorption. Ferrous (Fe²⁺) iron salts are absorbed better than ferric (Fe³⁺) salts, though absorption remains below 10% for both.

○ Absorption Enhancement: In the stomach, Fe³⁺ is reduced to Fe²⁺, which, when combined with Vitamin C, facilitates iron absorption.

○ Phytic acid, found in cereal husks, and substances like spinach and radish greens, bind to iron to form insoluble complexes, hindering absorption of irons.

○ Tannins in tea and coffee, as well as dietary fiber, also inhibit iron absorption.

○ Deficiency: Anemia

○ Cobalt (Co)

○ Copper (Cu)

○ Zinc (Zn)

○ Essential for the synthesis of proteins and collagen, necessary for growth, wound healing, and maintaining healthy skin.

○ Zinc is easily destroyed and is rarely consumed in sufficient amounts through food.

○ Abundant in seafood, red meat, nuts, beans, and milk.

○ Proteolytic enzymes require zinc ions for full activity.

○ Deficiency: Unlike iron, prolonged potential zinc deficiency does not immediately manifest symptoms.

○ Excess: Acute zinc toxicity.

○ Selenium (Se)

○ Acts as an antioxidant, preventing damage to cell structures.

○ Glutathione

○ Hydrogen Peroxide: A key component in its mechanism.

○ Iodine (I)

○ The thyroid is the only organ that absorbs iodine, a principle utilized in radiation therapy for thyroid cancer.

○ A component of thyroid hormones such as thyroxine, influencing fetal and child cell development and growth.

○ Contributes to white blood cells’ composition.

○ Moderately regulates milk secretion in nursing mothers.

○ Deficiency: Causes conditions such as thyroid hyperactivity (e.g., goiter, Graves’ disease (Basedow’s disease)), and cretinism.

③ Summary of Types of Minerals

Table 4. Summary of Types of Minerals

④ Intake

○ Minerals cannot be synthesized by the body.

○ Only a relatively small amount is required: Daily needs range from less than 1 mg to about 2500 mg.

○ As they are water-soluble, minerals can be lost during boiling.

⑻ Comparison of vitamins and minerals

① Vitamins are organic substances containing carbon, whereas minerals are not.

② Plants and bacteria can synthesize certain vitamins, but minerals cannot be synthesized.

③ Vitamins are easily destroyed by air, light, heat, etc., while minerals are chemically stable and not easily destroyed.

⑼ Phytochemicals: Bioactive compounds found in plants with strong physiological activity.

① Pigments: Have antioxidant effects, contributing to the prevention of aging and chronic diseases.

② Isoflavones: Found in soybeans, act similarly to female hormones, alleviating menopausal symptoms and preventing osteoporosis.

③ Garlic and onion: Contain substances that benefit heart health.

⑽ Calories and metabolic rate

① Calorie: The amount of energy required to raise the temperature of 1 g of water by 1°C. Kilocalories (kcal) are often abbreviated as “C.”

② Cellular energy use: Cells utilize nutrient energy for work and maintaining body temperature.

③ Metabolic rate: A measure of the speed of enzymatic reactions in the body.

○ Daily recommended intake: 2700 kcal/day for average adult male and 2100 kcal/day for average adult female.

○ Basic metabolic rate (75%): Energy expenditure at rest, influenced by thyroid hormones.

○ Activity metabolic rate (25%): Energy expenditure per hour required for specific activities.

④ Chemical reactions as energy sources:

○ C-C bonds: Long-term energy storage

○ C-H bonds: Long-term energy storage

○ C-OH bonds: Short-term energy storage

⑾ Nutritional imbalance

① Nutritional deficiency

○ Occurs when calorie intake is chronically insufficient to meet the body’s chemical energy needs.

○ Results in glycogen, fat, and protein breakdown, leading to muscle reduction and protein deficiencies in the brain.

○ Example 1. Sub-Saharan Africa: Approximately 200 million people lack adequate nutrition due to drought, war, or AIDS epidemics.

○ Example 2. Anorexia nervosa (compulsive fasting).

② Overnutrition

○ Occurs when animals consume more food than their energy needs, storing excess nutrients as glycogen or body fat.

○ Essential for hibernating animals.

③ Malnutrition

○ A condition caused by the deficiency of one or more essential nutrients.

○ Example 1. Vitamin A deficiency → Resolved by providing beta-carotene (e.g., golden rice).

○ Example 2. Herbivores eating plants grown in phosphorus-deficient soil → Weak bones.

○ Example 3. Diets lacking essential amino acids → Protein deficiency.

⑿ Evaluation of Nutritional Needs

① Using humans for research purposes raises ethical concerns.

② Hemochromatosis: A genetic disease in which iron accumulates even without abnormal iron consumption.

③ Epidemiological Research: Studies health and disease at the population level.

○ Example: Neural tube defects occur when the developing brain and spinal cord fail to close, but folic acid intake significantly reduces these defects during fetal development.

⒀ Acidic and alkaline foods

① Acidic foods

○ Definition: Foods that, when burned, leave ash rich in acidic elements such as phosphorus, sulfur, and chlorine.

○ Example: Egg yolks (high in phosphoproteins), fruits (due to citric acid and malic acid), foods that are major sources of energy and protein.

② Alkaline foods

○ Definition: Foods that, when burned, leave ash rich in alkaline elements such as calcium, potassium, sodium, and iron.

○ Example: Egg whites.

2. Enzyme

⑴ Metabolism: All chemical reactions in the body.

① Gibbs free energy: If ΔH - TΔS < 0, the reaction is spontaneous.

② In biology, ΔH is approximately considered equal to ΔG (ΔH ≒ ΔG).

○ Exothermic reaction: ΔH < 0, related to catabolism.

○ Endothermic reaction: ΔH > 0, related to anabolism.

③ Even if a reaction is spontaneous, the reaction rate can be slow if the activation energy (threshold energy) is too high.

④ Activation energy

○ Definition: Minimum energy required for reaction molecules to trigger chemical reactions.

○ Lower activation energy increases the reaction rate by increasing the number of molecules that can react.

○ Catalyst: Combines with the reactant substrate to lower the activation energy and increase the reaction rate.

○ Enzyme: Biocatalyst

⑵ Features

① Feature 1. Substrate specificity: Enzymes catalyze reactions by acting only on specific substrates that match the shape of their active site and three-dimensional structure.

○ Lock and key model: A model suggesting that the enzyme’s active site perfectly matches the substrate.

○ Induced Fit Model: A model suggesting that when an enzyme binds to a substrate, it changes into a completely complementary shape to fit the substrate.

○ 1^st. The shape of the substrate is roughly similar to the enzyme’s active site.

○ 2^nd. When the substrate binds to the active site, the enzyme changes shape and applies pressure on the chemical bonds.

○ 3^rd. This shape change breaks down the substrate and releases its monomers.

○ Isozyme (enzyme multiplicity)

○ Different enzymes involved in the same biochemical reaction.

○ Have different characteristics depending on the cell that acts (e.g., hexokinase, lactose dehydrogenase (LDH))

○ Each enzyme is regulated by feedback from its specific end product, resulting in different enzymes being active depending on the type of end product.

② Feature 2. Recycle: The amount of enzyme before and after the reaction is the same.

④ Feature 3. The enzyme only affects the reaction rate but does not affect the size of the reaction heat.

⑤ Feature 4. From common ancestors: When other organisms use the same enzymes.

⑵ Composition of enzymes

① Classification of enzyme: Divided into RNA enzyme (called ribozyme) and protein enzyme. Enzyme is commonly referred to as protein enzyme.

② Active site: Sites that bind to the substrate.

③ Holoenzyme: An enzyme that exhibits full activity.

④ Apoenzyme : Protein parts that make up the enzyme.

⑤ Cofactor: Nonprotein parts that make up enzymes. Attaches to the active site to complete the active site.

○ Coenzyme: Organic molecules required for the activity of the enzyme.

○ Example: Vitamin derivatives, NAD⁺, FAD

○ Inorganic ion: Metal elements such as Fe²⁺, Cu²⁺, Mg²⁺, Zn²⁺.

○ Prosthetic group: A type of cofactor that is strongly and permanently bound to an enzyme.

○ The porphyrin ring is a representative chemical structure that forms a prosthetic group, with the following examples:

○ Example 1. Hemoglobin heme group: An organic compound with Fe²⁺ contained in a porphyrin ring.

○ Example 2. Myoglobin

○ Example 3. Chlorophyll

○ Example 4. Cytochrome P450 (CYP)

⑥ Most hydrolytic enzymes, such as amylase, pepsin, and lipase, consist only of protein

⑶ Enzyme Catalytic Mechanism

① Acid-base catalysis

② Covalent catalysis

③ Metal ion catalysis

④ Electrostatic catalysis

⑤ Proximity and orientation effects

⑥ Preferential binding of the transition state complex

⑷ Factors affecting the action of enzymes

① Factor 1. Substrate concentration

○ Related to Michaelis-Menten equation

○ Initial reaction rate increases with increasing substrate concentration and then becomes constant after reaching a certain level.

○ Once all the enzymes are saturated with the substrate, the initial reaction rate no longer increases with increasing substrate concentration.

② Factor 2. Temperature

○ As temperature increases, the number of molecules with kinetic energy above the activation energy increases, increasing the reaction rate.

○ The chemical reaction involving enzymes is the fastest at the optimum temperature at which the enzyme has the optimum structure.

○ If the temperature exceeds the optimal level, the protein undergoes irreversible denaturation due to heat and cannot recover even if the temperature is reduced.

③ Factor 3. pH

○ The chemical reaction involving enzymes is the fastest at the optimum pH.

○ The charged state of the amino acid residues that make up the enzyme depends on the change in the concentration of hydrogen ions, so if it is out of optimum pH, it changes the net charge of the protein, causing electrostatic repulsion and altering the structure of the enzyme.

○ Examples of optimum pH

○ Pepsin: pH = 1.5

○ Catalace: pH = 7.6

○ Trypsin: pH = 7.7

○ Fumarase: pH = 7.8

○ Ribonucleace: pH = 7.8

○ Arginase: pH = 9.7

④ Enzyme reaction rate index

○ 1 unit of enzyme: Enzyme activity that can produce 1 μmol of product in 1 minute.

○ Enzyme activity = enzyme unit / amount of enzyme (ml)

⑸ Classification of enzymes: Defined by IUPAC. Also known as enzyme commission number.

① EC1: Oxidoreductase

○ A redox-mediated enzyme that transports hydrogen, oxygen, and electrons.

○ Type 1. “Reactant + dehydrogenase” (e.g., lactose dehydrogenase, alcohol dehydrogenase)

② EC2: Transferase

○ Transfer functional groups such as methyl, acyl and amino groups to other substances.

○ Type 1. “trans + reactant + -ase” (e.g., transaminase, acetyltransferase)

○ Type 2. “Reactant + -kinase” (e.g., hexosekinase)

③ EC3: Hydrolase

○ Participate in hydrolysis and condensation.

○ Type 1. “Reactant + -ase” (e.g., protease, peptidase)

④ EC4: Lyase

○ Catalyze the addition or removal of atomic groups by cleaving C-C, C-O, C-N, C-S bonds, etc. of the substrate.

○ Addition reactions involve two-substrate reactions, while elimination reactions involve single-substrate reactions.

○ EC4.4: Carbon-nitrogen bond cleaving enzyme

○ EC4.5: Carbon-halogen bond cleaving enzyme

○ EC4.6: Phosphorus-oxygen bond cleaving enzyme

○ Type 1. “Reactant + decarboxylase” (e.g., pyruvate decarboxylase)

⑤ EC5: Isomerase

○ Enzymes that rearrange the structure of a substance.

○ Type 1. “Reactant + isomerase” (e.g., phosphoglucose isomerase)

○ Type 2. “Reactant + mutase”

⑥ EC6: Ligase

○ ATP is used to form new bonds between two substances.

○ Type 1. “Reactant + ligase” (e.g., DNA ligase)

⑹ Michaelis-Menten equation

⑺ Inhibitor: In living organisms, feedback inhibition mechanisms exist to regulate enzymes, but unlike inhibitors, the reactions are reversible.

① Irreversible inhibition

○ Example 1. Penicillin

○ A bacterium cell wall (peptidoglycan) transpeptidase inhibitor, potent because it has a semi-permanent covalent bond with the active site

○ Highly effective due to its semi-permanent covalent binding to the active site.

○ Example 2. Sarin Gas

○ Acetylcholine is broken down into choline and acetic acid. Acetic acid dissociates into acetate ions and H⁺.

○ 1^st. Sarin gas irreversibly binds to the active site of acetylcholinesterase.

○ 2^nd. Acetylcholine levels increase (↑).

○ 3^rd. Results in muscle spasms, pupil constriction, confusion, and respiratory distress.

② Reversible inhibition

○ Classified into competitive inhibition, uncompetitive inhibition, and noncompetitive inhibition.

○ When discussing competitive, uncompetitive, or noncompetitive inhibition, the distinction between reversible and irreversible inhibition is often not emphasized.

③ Competitive inhibition

○ Competes with the substrate for the same active site, inhibiting the enzymatic reaction.

○ Michaelis-Menten equation

○ Does not alter the shape of the active enzyme.

○ Example

○ Ibuprofen: Inhibits prostaglandin production.

○ Malonic Acid-Succinate Dehydrogenase: Succinate dehydrogenase oxidizes succinate. However, malonic acid, a competitive inhibitor of succinate, binds to the enzyme’s active site, preventing the oxidation of succinate.

○ Statin-HMG-CoA reductase

○ Ras phosphorylation of Gleevec-Bcl-abr

○ HIV protease inhibitors

○ Antidepressants, antibiotics, and insecticides

④ Uncompetitive inhibition

○ An inhibitor binds to the enzyme-substrate complex, inhibiting the enzymatic reaction.

○ Michaelis-Menten equation

⑤ Noncompetitive inhibitors

○ Inhibits the enzymatic reaction by binding to an allosteric site other than the active site, resulting in noncompetitive participation in the enzymatic reaction.

○ Michaelis-Menten equation

○ Changes the form of active enzymes.

⑻ Cooperative

① Regulation of enzyme activity by the substrate itself, rather than inhibitors or activators.

② Positive cooperativity: In multi-substrate enzymes, the binding of one substrate facilitates the binding of additional substrates.

③ Negative cooperativity: Exists but is extremely rare.

④ Reaction Formula: In the presence of cooperativity, once ES₁ is formed, ES_n is rapidly generated, so there is no need to consider intermediate states such as ES₁, …, ES_n-1.

⑤ Example: Hemoglobin oxygen saturation

○ Hemoglobin consists of four subunits, each with an oxygen-binding site.

○ Hemoglobin exhibits an oxygen saturation curve with a sigmoid shape.

○ When one oxygen molecule binds to a site, the oxygen affinity of the remaining binding sites increases.

○ However, in regions with low oxygen, once one oxygen molecule dissociates, the rest are also likely to dissociate.

○ Myoglobin, with a single subunit, lacks cooperativity and therefore shows a hyperbolic oxygen saturation curve.

⑼ Enzyme activity regulation mechanisms

① Regulation by suppressor

② Regulation by cooperativity

③ Allosteric control

○ Regulatory molecule: A small molecule that binds to a protein, altering its three-dimensional structure and thereby changing its function.

○ Regulatory molecules are not substrates.

○ Regulatory molecules include inhibitors or activators.

○ Allosteric regulation

○ Refers to the binding of activators or inhibitors to sites other than the substrate binding site, modulating the enzyme’s activity.

○ These alternative binding sites are called allosteric sites.

○ Allosteric regulation occurs in enzymes involved in irreversible reactions.

○ Example: ATP acts as an inhibitor, and ADP as an activator, to regulate enzymes involved in cellular respiration.

○ PFK-1 (Phosphofructokinase-1) is an enzyme that catalyzes the conversion of F-6-ⓟ (fructose-6-phosphate) to F-1,6-bisphosphate.

○ ATP, as a substrate of PFK-1, also serves as an allosteric regulator of the enzyme.

○ A graph plotting ATP concentration on the x-axis and PFK-1 activity on the y-axis forms a bell-shaped curve.

○ Application 1. Inhibitors

○ General allosteric regulation does not completely block reactions as inhibitors do.

○ The binding of an inhibitor to an enzyme, triggering allosteric regulation, is referred to as noncompetitive inhibition.

○ Competitive inhibitors bind to the active site, where the substrate attaches, rather than to a different site, and thus are not considered allosteric regulation.

○ Application 2. Feedback Inhibition

○ Refers to a mechanism where the final product of a metabolic pathway binds to an enzyme involved in the initial steps, suppressing the pathway and regulating metabolism.

④ Regulation through phosphorylation

⑤ Regulatory proteins

○ Example: NO synthase regulation of Ca²⁺-Calmodulin

⑽ Enzyme immobilization: Immobilizing enzymes in specific positions

① Entrapment (encapsulation): Most widely used as physical enzyme immobilization method.

○ Porous hollow fiber

○ Spun fiber

○ Gel matrix

○ Micro-capsule

② Chemical bonding: A chemical enzyme immobilization method that uses chemical forces between the functional group of an enzyme and the surface functional group of a carrier to fix the enzyme.

③ Effective factor = Reaction rate with diffusion limitation / Reaction rate without diffusion limitation

○ How much better the reaction rate was by immobilizing the enzyme.

○ The higher enzyme concentration, the lower effective factor: Meaning that the reaction occurs well because the concentration is high regardless of fixation.

⑽ Enzyme Assay

① UV spectrometer

○ 260 nm spectrometer: Measure the absorbance of the nitrogen base of the nucleic acid.

○ 280 nm spectrometer: Most typically used, measuring the absorbance of phenyl groups of Phe, Trp, Tyr.

○ 340 nm spectrometer: NADH absorbance measurement.

○ 405 nm spectrometer: Ninhydrin reacts with amino acids to produce purple products of 570 nm absorbance.

○ 455 nm spectrometer: β-carotene absorbance measurement.

○ 474 nm spectrometer: Lycopene absorbance measurement.

○ 500 nm spectrometer: Carotenoid absorbance measurement.

○ 560 nm spectrometer: Hemoglobin absorbance measurement.

○ 680 nm, 700 nm spectrometer: Chlorophyll absorbance measurement.

○ 840 nm, 870 nm spectromter: Absorption Measurement of Pigment Molecules of Photosynthetic bacteria.

② Bradford method: Determination of the change in wavelength caused by Coomassie Blue G dye binding to protein.

○ Advantages: Dosing is very fast and simple.

○ Disadvantages: The degree of binding of dyes to proteins varies.

○ Application: Pierce 660 nm protein assay

③ BCA Method (Bicinchoninic acid method): Measurement based on the reduction of copper ions by amide bonds in amino acids.

○ Currently the most frequently used protein quantification protocol.

○ Advantages: Minimal differences between proteins, excellent sensitivity

Figure 1. Sensitivity difference between the BCA method and the Bradford method

○ Disadvantages: Requires numerous preparation reagents, involves complex procedures, and can be interfered with by other reducing agents, copper chelators, or high-concentration buffers.

○ Examples of reducing agents: DTT, β-ME

○ Examples of copper chelators: EDTA, EGTA

○ Step 1. Cu²⁺ → Cu⁺

○ Reduction is primarily driven by cysteine, cystine, tyrosine, and tryptophan.

○ Unlike the Bradford method, the peptide backbone also participates in the reduction reaction, resulting in minimal differences between proteins.

○ Step 2. The reagent forms a complex with Cu⁺, resulting in a color change.

○ Apple-green Cu²⁺ transitions to the purple Cu⁺-BCA complex.

Figure 2. Reaction of Cu⁺ complex formation

○ Related protocols: Includes variations such as the biuret reaction, Lowry method, and Peterson method.

④ Fluorescent protein assay

○ Thermo Scientific Quanti-iT, Qubit and NanoOrange protein assay

○ NanoOrange Protein Quantitation Kit

○ CBQCA Protein Quantitation Kit

○ EZQ Protein Quantitation Kit

○ Fluorometer

○ Invitrogen Qubit Fluorometer

⑾ Example of enzyme

① Example 1. Glucose transporter (GLUT): Transports glucoses in cell Membranes.

○ Characteristic: Bidirectional. Only type D glucose can be transported by the glucose carrier. Passes through the cell membrane 12 times.

○ GLUT1: Found in all cells. K_m = 1 mM

○ GLUT2: Found in the liver, pancreatic β-cells, and small intestine. K_m = 10-20 mM. Low sensitivity due to high glucose in the first place.

○ GLUT3: Found in the brain and abundant in immune cells. K_m = 1 mM. High sensitivity due to the brain’s high energy demand.

○ GLUT4: Found in the muscle and fat. K_m = 5 to 10 mM. Conditionally expressed by insulin.

○ GLUT5: Found in the fructose carrier and small intestine.

○ GLUT and insulin secretion mechanism

Figure 3. GLUT and insulin secretion mechanism

○ 1^st. Increase in blood glucose concentration.

○ 2^nd. Glucose enters pancreatic β-cells via GLUT2.

○ 3^rd. Glucose undergoes glycolysis and the TCA cycle, producing large amounts of ATP. Glucokinase acts as a glucose sensor.

○ 4^th. The generated ATP blocks ATP-sensitive K⁺ channels, increasing cation content inside the cell.

○ 5^th. The cell membrane depolarizes, opening Ca²⁺ channels.

○ 6^th. Ca²⁺ enters the β-cells and promotes the release of insulin vesicles, increasing insulin levels in the bloodstream.

○ 7^th. Insulin enters muscle cells and promotes the translocation of GLUT4 vesicles to the cell membrane.

○ 8^th. GLUT4 from the GLUT4 vesicles is expressed on the muscle cell membrane.

○ 9^th. GLUT4, being more sensitive than GLUT2, facilitates glucose uptake into muscles and fat, lowering blood glucose levels.

② Example 2. Sucrose Hydrolase

○ A hydrolytic enzyme that breaks down sucrose into glucose and fructose.

○ Monomeric structure with only an active site, lacking an allosteric structure.

③ Example 3. Lactose dehydrogenase (LDH)

○ Consists of 4 units of H (heat form) or M (muscle form).

○ A total of five types: H₄, H₃M, H₂M₂, HM₃, M₄

○ Isoelectric point of H is 5.7, and that of M is 8.4.

○ Muscle: Pyruvate → Lactate by muscle form LDH (∵ Muscle undergoes lactic acid fermentation).

○ Liver: Lactate → Pyruvate by muscle form LDH (∵ Liver performs gluconeogenesis).

○ Heart: Lactate → Pyruvate by heart form LDH (∵ The heart uses lactate as an energy source).

④ Example 4. Hexose kinase

○ Hexokinase I: Found in muscles, with very high substrate affinity for glucose.

○ Hexokinase IV: Found in the liver, with low substrate affinity for glucose.

⑤ Example 5. Ethanol decomposition

Figure 4. Ethanol decomposition

⑥ Example 6. Lactose intolerance

○ In infants, lactase is well secreted, but its production decreases in adulthood.

○ In European countries, lactose intolerance is less commonly observed.

○ Process: Lactase deficiency in the small intestine → inability to digest lactose → bacteria ferment lactose → causes gas and diarrhea.

Figure 5. Lactose intolerance

3. Transport through membrane

⑴ Component 1. Cell membrane: Follows fluid mosaic models.

⑵ Component 2. Membrane protein

① Overview

○ Membrane proteins that penetrate the hydrophobic interior of the cell membrane.

○ Over 50% of drugs administered to humans target membrane proteins.

② Type 1. Integral membrane protein

○ Transmembrane protein. Membrane penetration is alpha helix structure

○ Feature 1: The transmembrane region adopts an alpha-helical structure.

○ Feature 2: Proline and glycine cannot form alpha helices and thus cannot exist in the transmembrane region.

○ Examples: Channel proteins, carrier proteins, pumps

○ Functions: Enzymatic reactions (often protruding on only one side), Signal transduction

③ Type 1-1. Transport proteins: Responsible for transporting substances.

○ Classification 1: Based on the transport mechanism: Carrier proteins, channel proteins, pump proteins (see below).

○ Classification 2: Based on energy usage

○ Passive transport and active transport

○ If the transported molecule is very large, it is not classified as active transport but referred to as endocytosis or exocytosis.

○ Classification based on cotransport

○ Single-molecule transport: Aquaporins, glucose transporters (GluT), etc.

○ Symporters (same-direction cotransporters): Na⁺-glucose cotransporter, H⁺-sucrose cotransporter, etc.

○ Antiporters (opposite-direction cotransporters): Na⁺-H⁺ exchanger, Na⁺-Ca²⁺ exchanger, G3P-pi exchanger, etc.

④ Type 1-2. Integrins: Interact with the extracellular matrix (ECM).

⑤ Type 2. Peripheral membrane protein

○ Attachment to the cell membrane: Adheres to the membrane through electrostatic interactions.

○ Presence of glycoproteins: Glycoproteins are attached.

⑥ Hydropathy plot: Identify transmembrane areas.

⑦ Simple separation experiments of integral membrane protein (membrane fraction) and peripheral membrane protein (soluble fraction)

○ 1^st. Partial disruption of the cell membrane using physical methods (e.g., sonication).

○ 2^nd. Centrifugation to separate components.

○ 3^rd. The supernatant contains soluble proteins.

○ 4^th. Adding a surfactant (e.g., detergent) to the pellet allows the extraction of membrane proteins.

⑧ Extraction of Membrane Proteins

○ Integral membrane proteins: Extracted using detergents such as SDS or Triton X-100, disrupting the cell membrane.

○ Peripheral menbrane proteins: Extracted by altering pH, applying heat, or using NaCl or urea, increasing hydrophilicity.

○ Urea: Due to its strong hydrophilicity, urea removes all R-R interactions except for disulfide bonds.

⑶ Transport Mechanism 1. Passive Transport: Passive transport involves diffusion along the concentration gradient without using energy.

① Simple diffusion: Movement of molecules from high concentration to low concentration (faster when the concentration gradient is steeper).

○ Transport of small hydrophobic molecules through the phospholipid bilayer.

○ Does not require energy.

○ Continues until equilibrium is reached.

② Facilitated Diffusion: Diffusion of hydrophilic and charged substances through membrane proteins.

○ Channel Proteins (pore proteins)

○ Definition: Proteins that form a channel to transport water molecules or hydrophilic solutes via facilitated diffusion.

○ Ion-selective: Allows only specific ions to pass through.

○ Exhibits behavior similar to simple diffusion and does not saturate.

○ Classification 1. Dependency-based channels

○ Ligand-gated channels: Activated by ligands, e.g., acetylcholine receptors.

○ Voltage-gated channels: Activated by changes in membrane potential, e.g., action potential channels.

○ Mechanically-gated channels: Activated by mechanical stimuli, e.g., auditory hair cells.

○ Classification 2. Ion Channels (Ionophores): Rapidly increase the permeability of specific inorganic ions through the cell membrane.

○ Examples: H⁺ ion channel (DNP), K⁺ ion channel (valinomycin)

○ Carrier Proteins (porter proteins)

○ Definition: Proteins that change their structure to transport specific solutes via facilitated diffusion.

○ Transport is more than 1,000 times slower than channel proteins.

○ Follows Michaelis-Menten kinetics, can saturate similar to enzymes.

Figure 6. Mechanisms of carrier proteins

○ Water: The only molecule transported via both ion channels and carrier proteins.

③ Osmosis: The phenomenon in which free H₂O diffuses from a high concentration (low salt solution) to a low concentration (high salt solution) across a semipermeable membrane that only allows water to pass.

○ Formula

○ Hypotonic solution: A solution with a lower osmotic concentration than inside the cell. Causes hemolysis in animal cells and is the natural environment for turgidity in plant cells.

○ Plant cells and hypotonic solution

○ Water Potential = Osmotic Pressure - Turgor Pressure (provided that Turgor Pressure ≥ 0)

○ Condition for Water Potential = 0 or Turgor Pressure = Osmotic Pressure: Turgidity

○ Isotonic Solution: A solution with the same osmotic concentration as inside the cell. Provides a stable environment for animal cells but causes wilting in plant cells.

○ Osmotic concentration inside the cell = 0.9% = 0.3 M = 300 mOsmol

○ Hypertonic solution: A solution with a higher osmotic concentration than inside the cell. Causes cell shrinkage in animal cells and plasmolysis in plant cells.

○ Aquaporins: Structurally classified as channels but, unlike general channel proteins, can become saturated.

⑷ Transport Mechanism 2. Active Transport: Transport proteins move specific ions and molecules against their electrochemical concentration gradient.

① Characteristic

○ Involvement of carrier proteins called pumps: Active transport does not occur in a channel form.

○ Utilizes phosphorylation by ATP or excitation by light energy

○ Unidirectional transport

② Pump: Transport proteins involved in active transport. Divided into P type, V type and F type

③ P-type pump: Transports substances by hydrolyzing ATP, attaching a phosphate group to the pump, and inducing a structural change in the pump.

○ Na⁺ / K⁺ pump: Only present in animals. Pump 3 Na⁺ molecules out of cells and 2 K⁺ molecules into cells.

○ Example: When glucose is absorbed in the small intestine.

○ K⁺ pump: Present in plants, bacteria and fungi. Pumping hydrophilic nutrients such as sugar.

○ H⁺ Pump (P type): Present in plants, bacteria and fungi. Symport of sugar and lactose.

○ H⁺ / K⁺ pump: Involved in hydrochloric acid secretion by parietal cells in the stomach wall. Cl^- is transported via passive transport.

○ Ca²⁺ pump: Used for calcium storage in the smooth endoplasmic reticulum.

④ V-type pump: Directly utilizes the energy generated from ATP hydrolysis.

○ Vacuolar membrane proton pump

○ H⁺ pump (Type V): Creates an acidic environment inside lysosomes and vacuoles.

○ Inhibitors: Bafilomycin

⑤ F-type pump: ATP synthase acts as a pump to move H⁺ ions.

○ Bacteriorhodopsin

○ Performs light-dependent transport.

○ In bacteria and archaea, H⁺ ions are pumped upon exposure to light.

○ C-P-Q (carotene-porphyrin-naphthoquinone): A proton pump isolated from bacteria.

⑥ Direct active transport (primary active transport): Transport that directly utilizes energy for substance movement.

⑦ Indirect active transport (secondary active transport): Transport that utilizes the active transport of another substance.

⑧ Agents

○ Fusicoccin: Activates proton pumps.

○ Vanadate: Inhibits proton pumps.

⑸ Transport Mechanism 3. Endocytosis and exocytosis: Large molecules cannot be transported via passive or active transport.

① Characteristics: Utilizes ATP.

② Type 1. Exocytosis: Vesicles enclosed by membranes fuse with the cell membrane and release large molecules.

○ Example: Insulin. Insulin is not secreted through facilitated diffusion.

③ Type 2. Endocytosis: A vesicle forms around the large molecule, transporting it into the cell. Classified into categories ④–⑥.

○ Example: Iron in the blood binds with the transferrin protein carrier for endocytosis.

○ Excess cytochalasin D inhibits endocytosis by preventing actin polymerization.

④ 2-1. Phagocytosis: Actively absorbs material using pseudopodia.

○ Small amounts of cytochalasin D (10 μM) can inhibit phagocytosis.

⑤ 2-2. Pinocytosis: Involves random absorption by invagination of specific areas of the cell membrane.

○ Sometimes includes receptor-mediated endocytosis as part of pinocytosis.

○ Macropinocytosis

○ A significant characteristic of cancer cells.

○ Mechanism: Promoted by the RAS pathway, shown to contribute to drug uptake in cancer cells (ref).

○ Function 1. Enhanced under nutrient or growth factor deficiency to utilize extracellular substances for glutamine production.

○ Function 2. Overexpression of macropinocytosis via RAS can lead to methuosis, a form of non-apoptotic cell death (ref).

○ Signaling molecules: CTBP1, Rac1, Rabankyrin-5.

○ Inhibitor: EIPA (5-(N-ethyl-N-isopropyl)amiloride).

⑥ 2-3. Receptor-mediated endocytosis

○ Selectively absorbs substances by forming vesicles with receptors bound to substrates.

○ Example 1. Clathrin-mediated endocytosis (CME): Includes LDL receptor-mediated endocytosis.

○ 1^st. After LDL binds to its receptor, it is enclosed in a vesicle through receptor-mediated endocytosis and transported into the cell.

○ 2^nd. Adaptin, clathrin-coated pits, and dynamin are involved in the membrane invagination.

○ Clathrin: A protein that coats vesicle membranes during the endocytosis process. Forms a layer.

○ Dynamin: A GTPase involved in vesicle formation, responsible for severing the vesicle from the membrane.

○ Uncoating: The removal of adaptin and clathrin from the vesicle.

○ 3^rd. The receptor vesicle merges with early endosomes.

○ 4^th. LDL separates from the receptor, moving into early endosomes.

○ 5^th. Recycling: The receptor vesicle separates and moves back to the cell membrane.

○ 6^th. The early endosome, separated from the receptor vesicle, merges with a primary lysosomal vesicle derived from the Golgi apparatus.

○ 7^th. After fusion, it becomes a secondary lysosomal vesicle (mature endosome).

○ 8^th. The mature endosome merges with the mannose pathway to form a complete lysosome.

○ 9^th. Cholesterol is separated.

○ Inhibitors: Dynasore (dynamin inhibitor), chlorpromazine, pitstop 2

○ Example 2. Caveolar-mediated Endocytosis (CvME)

○ Forms vesicles approximately 60 nm in size.

○ Inhibitors: Dynasore, nystatin, filipin.

○ Example 3. Albumin pathway

○ 3-1. gp60-mediated endocytosis

○ 3-2. SPARC-mediated endocytosis

○ 3-3. gp18, gp30-mediated endocytosis

○ 3-4. Megalin/cubilin-mediated endocytosis

○ 3-5. FcRn-mediated endocytosis.

○ Example 4. Transcytosis: A vesicle formed through endocytosis crosses the membrane.

○ Example 5. B Cell Receptor

○ Immature B cells express IgD, which binds antigens for uptake and fragmentation via lysosomes.

○ Fragmented antigens are labeled to MHC class II in the rough ER, converting tertiary structures to primary structures.

○ Example 6. Fc receptor-mediated endocytosis

○ Inhibitor: Piceatanol

○ Example 7. Mannose receptor-mediated endocytosis

○ Inhibitor: Mannan

○ Example 8. Intestinal epithelial cells

○ Intestinal epithelial cells position IgA towards the intestinal lumen via transcytosis to eliminate bacteria.

○ General target receptors and ligands

Input : 2015.6.25 10:01

Modify : 2019.2.16 09:45

69

Chapter 3. Cell and Material Metabolism

1. Nutrient

2. Enzyme

3. Transport through membrane

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