MBI 105-106 Medical Biochemistry Biochemical Aspects PDF

Medical Biochemistry Department First year biochemistry module Biochemical Aspects I. Question 1 comparison Feature Reversible - Competitive Reversible - Non-competitive Inhibitor structure Similar to substrate Not similar to substrate...

Medical Biochemistry Department First year biochemistry module Biochemical Aspects I. Question 1 comparison Feature Reversible - Competitive Reversible - Non-competitive Inhibitor structure Similar to substrate Not similar to substrate Site of binding Active site Allosteric site E-I interaction E+I EI E+I EI, ES+I ESI Effect on Vmax Not affected Decreased Effect on Km Km increased Not affected Lineweaver-Burk Same point of uninhibited Same point of uninhibited plot reaction (intersection on y reaction (intersection on x axis) axis) Reversal of By increasing substrate conc. Not by increasing substrate inhibition conc. may be by dialysis Examples Sulfonamides, Methotrexate, Negative feedback inhibition Dicumarol. Michaelis-Menten Kinetics Lineweaver-Burk plot 1  Medical Biochemistry Department Point of difference absolute enzyme specificity Relative enzyme specificity Definition The enzyme acts on a single The enzyme acts on structurally substrate only. similar substrates, not limited to one. - Glucokinase act only on- Hexokinase acts on hexose Example glucose but not other sugar (glucose, fructose, mannose). hexoses. -Urease acts only on urea. Aspect Functional Plasma Enzymes Non-Functional Plasma Enzymes Concentration in Present in higher concentrations Normally present in very low Plasma concentrations Function Have known functions in plasma No known functions in plasma Substrates Their substrates are always present in Their substrates are absent from the the blood blood Site of Synthesis Liver Different organs (e.g., liver, heart, brain, skeletal muscles) Examples Clotting factors (e.g., prothrombin) ALT, AST, CK, LDH, Alkaline phosphatase, Acid phosphatase, Amylase Lipoprotein lipase, Alpha helix Beta pleated sheet Shape coiled zigzag No of amino acids 3.6 amino acids/turn 5-10 AA in the poly peptide chain Stabilized by hydrogen bond hydrogen bond No of polypeptide chain One polypeptide chain More than one lined side by side 2  Disruption by proline Yes no Levels of protein 2ry structure 2ry structure organization Examples Hemoglobin Beta keratin in silk and spider Myoglobin venom Alpha keratin Point of Glycogen Starch difference Structure - Branched chain formed of thousands - Branched chain formed of thousands of of D-glucose units linked together by D-glucose units linked together by alpha alpha 1,4 glucosidic bonds, at the 1,4 glucosidic bonds, at the branch branch points the bonds are alpha 1,6. points the bonds are alpha 1,6. - It resembles starch in structure, but more extensively branched. - Main storage form of glucose in - Main storage form of glucose in plants. humans. Site Present in liver and muscles. It is present only in plants. Point of difference Fats and Oli Wax Alcohol Alcohol is glycerol. Alcohol is high molecular weight monohydric alcohol. Structure Esters of fatty acids with glycerol. Esters of fatty acids with high molecular weight monohydric alcohol. 3  Medical Biochemistry Department Fibrous Proteins Globular Proteins Axial ratio More than 10 Not more than 4 i.e. the protein is compact and coiled Solubility Water insoluble Mostly water soluble Examples Structural proteins (Scleroproteins) Dynamic proteins e.g. Insulin, albumin, e.g. globulins, and enzymes. Collagen, elastin, and -keratin Point of Denaturation difference Point of difference Acidic amino acid Basic amino acid Contain Acidic side chain at the Contain basic side neutral pH. chains at the neutral pH. Charge Negative Positive example Aspartic acid and glutamic acid. Lysine,histidine and Arginine Polar or not polar polar Types of bonds formed Electrostatic bond Electrostatic bond (non- (non-covalent) covalent) 4  Point of difference Monomers Polymers Small, single molecules that can bind to other Large molecules composed of Definition identical molecules to form a polymer. repeating units (monomers) linked together. Examples Glucose, amino acids, nucleotides. Starch, proteins, DNA Formation By hyaliphaticdrolysis reaction By condensation reaction Point of difference OH containing amino acid Sulfur containing amino Acid Contain OH group. SH group. Charge uncharged Uncharged example Serine, threonine. Cysteine, methionine Polar or not Polar. Cysteine (polar) Methionine(non polar) Point of High biological value protein Low biological value protein difference Contain all the essential amino Lack one or more essential acids required for synthesis of amino acids. protein. Most plant protein (zein of Proteins of animal origin: meat, maize deficient in liver, milk, fish. tryptophan and lysine). Some plant proteins(beans, Non digestible lentils, potato)when used in scleroproteins. combination. Gelatin produced by boiling of collagen. Example Meat, liver, milk, fish, beans, lentils Gelatin, zein of maize, and potato. scleroproteins. 5  PCR Gene cloning gene in vitro gene amplification in vivo gene amplification amplification (millions of copies) steps denaturation (separation of DNA ,95 1-Isolation of DNA fragment by restriction C) endonucleases = Molecular scissors (Palindrome annealing (primer bind to DNA, 55c pattern) Extension (synthesis of DNA by Taq 2-Ligation to a vector (host DNA) polymerase (thermostable DNA 3-Transformation, moving recombinant DNA polymerase) from thermos aquatics bacteria, 70) from test tubes into host cell 4- Selection of host cells containing the recombinant DNA by testing some markers carried by recombinant DNA, e.g., antibiotic resistance gene (bacteria without recombinant plasmid will die) requirements Target DNA Target DNA Nucleotides (dNTPs) host cell DNA polymerase enzymes Primers vector purposes Diagnosis of diseases Therapeutic applications 6 Aspect Somatic Gene Therapy Germline Gene Therapy Target Cells Somatic cells Germ cells (sperm, eggs) Heritability Changes are not passed to offspring (non- Changes are passed to future generations heritable) (heritable) Procedure Genes introduced into bone marrow cells, Genes introduced into ova and sperms blood cells, skin cells Feature Balanced Cell Growth Unbalanced Cell Growth Definition Cell gain= cell loss Cell gain is more than cell loss Deregulation of growth control Strict regulatory mechanisms mechanisms, Regulation ensuring normal growth. due to genetic mutations or environmental factors. Normal cell proliferation Examples during tissue maintenance and Cancer cell growth repair. Regular, well-coordinated Cell Cycle Irregular cell cycle progression progression through the cell progression (uncontrolled division) cycle. 7  G1 S Checkpoint G2-M Checkpoint (Restriction point) Location Boundary: near the end Boundary:between G2 and M phase. of G1and before entry into S phase Regulate initiation of DNA Sense problem in DNA Function replication. replication , DNA repair. Point of Short term enzyme Long term enzyme difference regulation regulation Definition Enzyme catalytic activity (takes Enzyme quantity (takes hours to seconds to minutes) can rapidly days) change the enzyme from inactive to fully active form. 1- Allosteric regulation. MechanismS 2- Reversible covalent Enzyme aynthesis modification. 1- Induction 3- Proteolytic Cleavage. ( increase synthesis of enzyme). 4- Compartmentation. 2- Repression. Occurs by controlling the number or the concentration of enzyme molecules present, determined by the rate of synthesis and rate of degradation of the enzyme protein (enzyme turnover). Enzyme degradation 8 Question II: Explain the biochemical mechanisms of the following conditions/drugs Drugs\ Disease Biochemical basis Allopurinol in- Allopurinol act as suicide enzyme inhibitor, it is structurally similar to hypoxanthine. - It inhibits xanthine oxidase enzyme: treatment of gout - Allopurinol is a substrate for xanthine oxidase. Allopurinol is converted into an active metabolite alloxanthine that irreversibly inhibits xanthine oxidase, thus decreasing uric acid production. Alzheimer accumulation of abnormally folded protein in the brain called amyloid precursor Disease protein(APP) which is critical to neuron growth. This misfolded protein (beta amyloid) will then be degraded into smaller fragments called beta- diminished and xanthine amyloid which and hypoxanthine form plaques levels in the blood rise. around neurons. tau protein hyperphosphorylation forming neurofibrillary tangles that destabilize microtubules and impair neuronal function Neonatal Lung surfactant ( D i p a l m i t o y l l e c i t h i n ) respiratory decreases the surface tension in the lung alveoli. So it prevents distress adherence of the inner surfaces in the lung. In premature infants, the lungs may not produce enough surfactant, leading to Lactose -neonatal Lactoserespiratory intolerancedistress syndrome is a condition (NRDS). by the inability to digest lactose. characterized Intolerance - Symptoms: Cramps, diarrhea, flatulence that are caused by the inability to digest lactose (milk sugar). - Biochemical basis: - Lactose digestion requires the action of the enzyme lactase. - Lactase deficiency leading to inability to digest lactose, undigested lactose is metabolized by intestinal bacteria leading to the gases and short chain fatty acids that cause the listed symptoms. Muscle atrophy in Due to decreased intake and increased loss of proteins. Lack of essential dietary malnutrition requirements like essential amino acids and essential fatty acids which will cause - protein energy malnutrition. 9  Loss of Because of environmental heat exposure with lack of thermoregulation. consciousness in Heat/denatured proteins/cytotoxicity. Increased release of toxic metabolites. High heat stroke heat affects sensitive tissues of the brain, denaturing proteins and disrupting communications. End organ damage. Organ Lowering body temperature is crucial in medical procedures like organ cryopreservation transplantation. As the enzymatic activities slow down, which minimizes in organ metabolic processes and decreases the risk of tissue damage. thus helps preserving transplant organs in cryopreservation for transplantation, maintaining their viability. Pencillin as an Irreversibly inhibits transpeptidase, blocking cell wall synthesis in bacteria, efficient Causes bacterial cell lysis and death. antibiotics Aspirin as an Irreversibly inhibits cyclo-oxygenase COX enzymes, reducing prostaglandin and analgesic and thromboxane production. antithrombotic Reduces inflammation, pain, and blood clotting. Biochemical basis - Pancreas produces protein digestive enzymes as zymogens and they are activated in of pancreatitis the intestine. - If these enzymes are inappropriately activated within the pancreas, they will digest the Organophosphorus Irreversibly inhibitscellular pancreas causing acetylcholinesterase, preventing acetylcholine damage and inflammation of pancreas. breakdown. compounds Leads to continuous muscle stimulation, potentially causing paralysis and death. (insecticides) are poisons for humans Carbon monoxide isBinds irreversibly to cytochrome c oxidase, blocking electron transfer in the toxic to the tissues electron transport chain. Inhibits cellular respiration 10  Medical Biochemistry Department Cellulose as Cellulose is present in cell walls of plants. It is considered as dietary fibers, it has Dietary fibers a very important role in diet: 1- It prevents constipation by: (a) adding bulk to stool, so it stimulates the intestinal wall. (b) it can absorb 5-10 times as their own weight water, which is drained into intestinal lumen increasing bowel movement. 2- It binds to toxic compounds present in diet decreasing the absorption, so they protect against colon cancer.. antiphospholipids Phosphatidyl serine, inositol, glycerol and cardiolipin play an important role in syndrome blood coagulation and clot lysis. Antibodies against acidic phospholipids cause recurrent thrombosis and/or fetal loss. cholesterol gall When there is an excess of cholesterol relative to bile salts, it becomes supersaturated, stone leading to the precipitation of cholesterol crystals and formation of stones. Sulfonamides act as - Bacteria synthesize folic acid from p-aminobenzoic acid (PABA). antibacterial drugs - Sulfonilamide is an antibiotic that is structural analog of PABA, so it blocks the synthesis of folic acid which is essential for bacterial multiplication. Methotrexate acts as - It is structurally similar to folic acid.Competitively inhibit dihydrofolate anticancer drug reductase enzyme, inhibiting folic acid activation so inhibit DNA synthesis & cell division. Question III- Enumerate the biological functions of each of the following items: Item Biological Functions Sugar alcohol in Sugar alcohols are commonly used in place of table sugar (sucrose). artificial sweetener Of these, xylitol is perhaps the most popular due to its similarity to sucrose in appearance and sweetness. They contain fewer calories than sucrose. Sugar alcohols are not metabolized by oral bacteria, and so they do not contribute to tooth decay. Glutathione Glutathione is a tripeptide acting as antioxidant Essential fatty acids 1- Cell Membrane Structure 2- Role in synthesis of eicosanoic acids. 3- Cognitive Function 4- Cholesterol lowering effect 11 Allosteric modifiers - Allosteric modifier binds to the allosteric site on the enzyme, induces conformational changes of the enzyme that leads to either: - Activation of the enzyme by positive modifiers e.g. Substrate - Inhibition of the enzyme activity by negative modifier e.g. end product feedback inhibition. It makes the enzyme less suitable to the substrate. Allosteric enzymes Have one or more allosteric sites, do not follow michelis-menten kinetics Usually have more than one polypeptide chain. Reversible covalent 1- Phosphorylation /dephosphorylation (most common) by protein kinases modification and phosphatases 2- Methylation and demethylation 3- Acetylation and deacetylation Chaperones Folding accessory protein. Which helps in folding the protein and giving the protein its shape that will be able to function properly. And help in Refolding denatured proteins. Functions of 1- Cell Membrane Structure: They form the bilayer of cell membranes, providing Phospholipids structure and regulating substance movement. 2- Signal Transduction: Certain phospholipids participate in cell signaling pathways. 3- Emulsification: Phospholipids stabilize mixtures of water and fat, important in digestion and food production. 4- Lipid Transport: They are part of lipoproteins that transport lipids in the bloodstream. 5- Cholesterol solubilization: PL solubilizes cholesterol in bile, any defect lead to cholesterol stones 6- Special function of phospholipids (Lung surfactant) Importance of bile salt Bile salts and bile acids are synthesized from cholesterol. Emulsification: Bile salts break down large fat droplets into smaller ones, aiding fat digestion. Fat and Vitamin Absorption: They help absorb dietary fats and fat- soluble vitamins (A, D, E, K) in the intestines Cholesterol execretion: Bile salts assist in removing excess cholesterol from the body Cholesterol solubilization: Prevent precipitation of cholesterol in bile and prevent formation of cholesterol stones. 4- They stimulate liver cells to secrete more bile(choleretic effect). 12 IV-Outline the following Properties of amino Physical properties :1- colorless, 2- soluble in polar solvent/ less polar acids in alcohol/ non polar in non polar solvent, 3- sterioisomerism: Enantiomers are mirror images of each other, there are two enantiomers (L and D forms) for most amino acids( L-Alanine& D- Alanine) Chemical properties :amphoteric can act as acid or base , buffering activity (resist changes in PH) 1ry structure of protein Linear sequence of AA (Number- type- arrangement) that make up protein structure. It is determined by DNA sequence of its genes. The amino acid sequence specify the 3D shape of the protein. β- turns Β Turns connect alpha helical and / or beta pleated sheets regions of peptide chain together. Usually composed of 4 amino acid residues, the first amino acid being hydrogen-bonded to the fourth Contain glycine and proline Stabilized by hydrogen bonds and ionic bonds The principal driving The association of hydrophobic R groups so as to bury hydrophobic force of the folding amino acids in the interior of the protein. process Minimizing the contact of hydrophobic amino acids with the polar solvent (water). protein misfolding Misfolded protein (Amyloid beta sheet)accumulation disorders 1. Alzheimer disease 2. Parkisonism 3. prion diseases 4. Cystic fibrosis 5. α1-antitrypsin deficiency. Factors causing denaturation 13 Effect of denaturation Change of the physical properties: 1- Decreased solubility. 2- Increased viscosity. Change of the chemical properties: 1- Non covalent bonds are disrupted (hydrogen bonds, ionoc bonds, vandewal forces) 2- Covalent bonds are not affected. Change of the biological activity: 1- Loss of enzyme,hormonal,antigenic activity. 2- Denaturated proteins are insoluble and precipitate from solution. Properties of the They are All enzymes are proteins degradedexcept at a faster rate than native proteins. ribozymes. Enzymes act as catalysts, speeding up reactions Enzymes Enzymes are highly specific to their substrate and to their reaction. Neither consumed nor produced by the reaction, and can be used repeatedly. Some enzymes require a cofactor or coenzyme. Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need. \ Models for Enzyme Lock and key model (rigid): The active site has aspecific shape that exactly matches the shape of its substrate. active site mechanism of action Induced fit model (more flexible): Both the enzyme and the substrate surfactant undergo conformational changes upon binding to each other. Binding sites not fully formed 14 Biologically active they contain less than 50 amino acids that exert specific physiological peptides effects in the body e.g. glutathione, endorphin, bradykinin, vasopressin - glutathione: tripeptide, antioxidant - Endorphin: Natural pain killer - bradykinin: Blood Vs dilation so, decrease blood pressure - vassopressin: Blood Vs constriction so, Raise blood pressure, Regulate water balance in the body by increasing water reabsorption in kidneys Mechanisms of proto- - Point mutation oncogene activation - Gene amplificatipn - Chromosomal translocation - Insertional mutagenesis 15 The importance of Definition of Km: The substrate concentration at which the reaction rate is half Michaelis Constant maximal velocity. (Km) It measures relative affinity of an enzyme for its substrate (low Km means higher affinity of E to S , high Km means low affinity of E to S). Determining enzyme inhibition: Knowledge of 𝐾m is essential in studying enzyme inhibition. Km and Vmax can be affected by pH and temperature. - Increase in substrate concentration will increase the rate of the enzyme activity till a certain level at which V-max is reached, at which all active sites of enzymes are saturated with the substrate, with enzyme concentration kept constant. Factors that affect the Temperature. rate of PH. enzyme activity Concentration of substrate and enzyme. Cofactors. Effect of PH on Within the optimum range , pH changes the charged state of both enzyme and enzyme activity substrate: 1- The pH affects the ionic state of the enzyme and of the substrate. Slight Changes in pH lead to Change in the charge of substrate and in the enzyme active site decreasing the binding between them. Marked changes in pH (high or low pH value) lead to Enzyme denaturation and irreversible inhibition of enzyme action. Optimum pH of most enzymes is 5-9. Effect of temperature The rate of enzyme activity typically increases with temperature up to an on enzyme activity optimal point. Beyond the optimum temperature, the enzyme may denature, losing its structure and activity. The temperature increases the energy of the substrate to overcome the energy barrier of the reaction. The (optimum) temperature for the enzyme is the temperature at which the enzyme act by its full rate(37'C) maximum velocity is reached. Temperature coefficient = Q10,between 0'and40' C, the velocity of the enzyme doubles with a 10'C rise in temp. each 10'C increases activity by 2 folds. 16 Feed- Back Inhibition Simple reversible noncompetitive inhibition. of enzymes In cell metabolism, the end product of a specific biochemical pathway can inhibit the first enzyme of this pathway. Proenzymes - A zymogen, also called a proenzyme, is an inactive precursor of an (zymogens) enzyme. - A zymogen requires a biochemical change such as a hydrolysis reaction (proteolytic cleavage) revealing the active site, or changing the configuration to reveal the active site, to become an active enzyme. - It is irreversible reaction, only reversed by specific inhibitors. - Classical example: ▪ Digestive enzymes to protect the cells producing them e.g. pancreas from autodigestion. 17 Enzymes of diagnostic - Alanine Aminotransferase (ALT) & Aspartate Aminotransferase (AST): Liver Importance diseases - Alkaline Phosphatase (ALP): Liver disease, bone disorders, bile duct obstruction - Gamma-Glutamyl Transferase (GGT): Liver disease, alcohol abuse, bile duct obstruction - Creatine Kinase (CK): Muscle damage or injury, heart attack - Lactate Dehydrogenase (LDH): heart attack, liver disease, hemolytic anemia - Amylase& lipase: Pancreatitis, pancreatic disorders - Prostate-Specific Antigen (PSA): Prostate cancer Criteria and examples Definition: Of isoenzymes They are enzymes that catalyze the same chemical reaction. They are different molecular forms of the same enzyme synthesized by different tissues. They share: 1- Catalyzing the same reaction. 2- Acting on the same substrate. 3- Using the same coenzymes. They are different in: 1- Amino Acid Sequence 2- Tissue Distribution. 3- Optimal Conditions e.g., pH & Temperature. 4- Electrophoretic mobility. Examples: 1- Lactate dehydrogenase. 2- Creatine kinase. (CK1 in brain , CK2 in heart, CK3 in skeletal muscle) Some enzymes are Streptokinase: used as Therapeutic an enzyme used as a therapeutic agent to dissolve blood clots in patients with agents conditions like myocardial infarction (heart attack) It works by converting plasminogen, a precursor in the blood, into plasmin, an active enzyme that breaks down fibrin, the main component of blood clots. Asparaginase: an enzyme used to treat acute lymphoblastic leukemia (ALL) by depleting asparagine, an amino acid essential for cancer cell survival, leading to the death of leukemic cells. Monosachrides Sugar acid: like Gluconic acid, glucuronic acid. derivatives Deoxy sugars: like 2 deoxy-ribose Amino sugars : Glucosamine and galactosamine Sugar alcohols: Sorbitol (Alcohol of glucose) Mannitol: Alcohol of mannose / xylitol: alcohol of xylose Xylitol: Alcohol of xylose 18  Importance of dietary - They prevent constipation by: (a) They add bulk to stool, so stimulate Fibers intestinal wall. (b) They can absorb 5-10 times as their own weight water, which is drained into intestinal lumen increasing bowel movement. - They protect against cancer: They bind to toxic compounds present in diet decreasing their absorption. Importance of Cholesterol - Cholesterol enters in the structure of the cell membrane - It is the precursor of bile salts, steroid Hormones and vitamin D3. Comparing CKMB (CK2) and LDH1 in acute myocardial diagnosis The cardiac isoenzymes of both CK & LDH have increased. CK2 rises within 4-8 hours after the onset of pain reaches peak within 24 hours. It returns to normal level after 2-3 days. LDH1 rises within 24-48 hours, reaches its peak after 2-3 days. It returns to normal level after 8-14 days. CK2 is used for early detection (recent infarction) LDH1 for follow up 19  Medical Biochemistry Department Identify the provided figure regarding protein structure 1. Primary structure: linear amino acid sequence stabilized by peptide bond 2. Secondary structure: Folding of a single chain into alpha helix and beta sheet, stabilized by hydrogen bond 3. Tertiary structure: Three-dimensional structure of protein, stabilized by covalent & noncovalent bonds 4. Quaternary structure: Arrangement of multiple protein subunits in space stabilized by non-covalent bonds 5. Beta turns stabilized by hydrogen & ionic bonds Define protein folding and its mechanism? Definition : the process by which a protein folds into its native, functional three-dessentialimensional structure. Mechanism : 1) Spontaneously As they are synthesized inside the cell. 2) Assisted folding By 1-chaperones(first discovered in bacteria in response to heating) 2-disulfide isomerase -Cis-trans isomerase 20  Remember AA Classification Aliphatic: AA with hydrocarbon side chain : (glycine/ alanine) - branched (valine , leucine, isoleucine) ▪ Side chain containing OH group(serine, threonine) ▪ Side chain containing sulfur group (cysteine, methionine) Note: Cysteine is involved in formation of covalent disulfide bond between polypeptide Chemical chains to stabilize protein structure. classification ▪ Side chain containing acidic groups (aspartic , glutamic) and amide derivatives (based on (aspargine- glutamine) structure of side ▪ Side chain containing basic group ( arginine, lysine, histidine) chain) ▪ Note: Ionic bond is formed between oppositely charged amino acids(acidic and basic amnio acids) Cyclic ▪ Aromatic (phenyl alanine, tyrosine, tryptophan(largest)) Note: (tyrosine and tryptophan absorb UV light at wave length 280 ) ▪ Heterocyclic (proline (imino acid) , histidine (basic) A) Polar: can reacts with water Polar charged ( present on surface of protein) ▪ Positively charged (Basic AA) ( arginine, lysine, histidine) ▪ Negatively charged(Acidic AA) (aspartic acid , glutamic acid) Polar uncharged ▪ AA containing OH ▪ AA containing SH Polarity of R ▪ AA containing amide Non polar can’t react with water( present in the interior of the protein): ▪ Aliphatic side chain(glycine smallest, achiral, alanine) ▪ branched (valine , leucine, isoleucine) ▪ Sulfur containing (Methionine) ▪ Aromatic: Phenylalanine- tryptophan ▪ Proline Essential (not synthesized inside body and must be supplied in diet) valine, leucine, isoleucine, threonine, methionine, lysine, phenylalanine, tryptophan. Nutritional Non- essential(can be synthesized) semi essential (Arginine , histidine) (in children) Metabolic Fate of AA in body (glucogenic, ketogenic , mixed) classification 21  Amino acids There are 20 AA derived from human protein Formed of alpha carbon connected to : Amino group peptide Radical group ( unique for each AA) H group Peptide bonds A peptide bond is a covalent chemical bond formed by dehydration or condensation reaction that linking the carboxyl group of one free amino acid molecule to the amino group of another. When three amino acids are linked together (two peptide bonds), a tripeptide is produced. Ex glutathione Given the following chain of amino acids: Val–Cys–Asp–Leu–Ala–Arg–Phe–Glu–Trp Identify the type of this peptide? (9 amino acid= nanopeptide, 8 peptide bond) identify amino (N)terminal? Val identify carboxyl (C) Terminal? Trp Remember carbohydrates classification: Monosaccharides Disaccharides (2) Polysaccharides Triose Lactose: (milk sugar) 1. Homopolysaccharides (starch and glycogen, Aldotriose: Formed of D-glucose and D-galactose cellulose glyceraldhyde Tetrose Sucrose: (Cane sugar or Table sugar Starch: present only in plants. Aldoterose: or sugar of beets)) -Formed of thousands of D-glucose units erythrose -Formed of D-glucose and D-fructose Glycogen: Main storage form of glucose in humans -Non reducing Pentose Maltose Cellulose: Formed of D-glucose Ribose Formed of D-glucose and D-glucose - Cellulose cannot be digested by the gastrointestinal enzymes of humans. - Cellulose present in cell walls of plants. - They are called Dietary fibers - Cellulose prevents constipation Hexose Disaccharides are connected by 2. Heteropolysaccharides (glycoprotein) Aldohexose: glucose glycosidic bond Ketohexose: fructose 22  Monosaccharides According to Functional number of carbons group Triose (3C) Tetrose(4C) Pentose (5C) Hexose (6C) Aldehyde ketose Glyceraldhyde Ribose Glucose, Erythrose glucose fructose galactose (aldotriose) (Aldotetrose) (Aldopentose) (Aldohexose) Dihydroxyaceton Erythroulose Ribulose Fructose e(ketotriose) (Ketopentose) (ketoterose) (ketohexose) Remember lipid classification Simple lipid Compound lipid Derived lipid 1. Triglycerides (Neutral - These are esters of FA with alcohol containing Result from fat) additional[prosthetic] groups. hydrolysis of o Alcohol is GLYCEROL - Biological membranes are made up of simple or o Esters of fatty acids with phospholipids compound lipids glycerol. 2. Waxes (Beeswax) o 1-Pohospholipids - Fatty acid o Alcohol is other than -Phosphatidic acid is the precursor of all - Alcohol glycerol phospholipids containing glycerol. - Steroids o Esters of fatty acids with o 2-Glycolipids (Cholesterol and bile high molecular weight Phospholipids: are classified according to type of alcohol acids) monohydric alcohols. A- Glycerophospholipids (Alcohol is glycerol): B- Sphingomyelin 1- Phosphatidic acid (Alcohol is sphingosine) 2- Phosphatidyl serine 3- Phosphatidyl ehanolamine 4- Phosphatidyl choline 5- Phosphatidyl inositol 23 Remember classification of fatty acids Enzymes are proteins except for certain classes of RNA (ribozymes) which exhibit catalytic activity Enzymes act as a biological catalyst by lowering the energy barrier of these reactions required for cell survival. 24  A-Enzyme synthesis I-Long term regulation Enzyme regulation (Enzyme quantity) B-Enzyme degradation A-Allosteric regulation B-Reversible covalent modification II-Short term regulation (Enzyme catalytic activity C- Proteolytic cleavage (Proenzymes) D- Compartmentation 25 Describe the Basic Steps of gene cloning 1-Isolation of The DNA fragment to be cloned needs by restriction endonucleases (Palindrome pattern) 2-Ligation of DNA fragment to a vector (host DNA) 3- Transformation, insertion of recombinant DNA into bacteria (or host cell) 4- Selection of the transformed cell by testing some markers carried by recombinant DNA, e.g., antibiotic resistance gene (bacteria without recombinant plasmid will die) List types of vector: Vector is a molecule of DNA that carry the gene to be cloned Prokaryotic Plasmids (bacteria) Viruses Cosmid Applications of Gene Cloning: 1. Gene Function Studies: Understanding the role of specific genes in biological processes and disease mechanisms. 2. Recombinant Protein Production: Examples include insulin, growth hormones, and enzymes. 3. VaccineDevelopment:Producingantigensforvaccines,suchasthoseusedinhepatitis B and human papillomavirus (HPV) vaccines. 26 Polymerase chain reaction (PCR): List PCR steps 1. Denaturation (separation of DNA ,95 C) 2. Annealing (primer bind to DNA, 55c) 3. Extension (synthesis of DNA by Taq polymerase from thermos aquatus bacteria, 70) At the end of a cycle, each piece of DNA in the vial has been duplicated Taq polymerase elongate DNA strand by adding new nucleotides at DNA primer PCR Applications 1. Genetic Research: Amplifying specific DNA sequences for studying gene function, structure, and variation. 2. Medical Diagnostics: Detecting genetic disorders, pathogens (e.g., bacteria, viruses), and mutations associated with diseases. 3. ForensicAnalysis:Identifyingindividualsincriminalinvestigationsandpaternity testing through DNA profiling. 27  Compare between different phases of cell cycle Describe cell cycle control at G1-S checkpoint. 28 Describe the general steps of CRISPR 1- Recognition: The process begins with the design of a single guide RNA (sgRNA). This sgRNA recognize a specific target complementary DNA sequence, forming a base pair. 2- Cleavage: The Cas9 nuclease cuts the DNA double strands (double-strand breaks (DSBs)) at the precise location specified by the sgRNA. 3- DNA Repair: Repairing the DNA break through natural cellular processes like non- homologous end joining (NHEJ) or homology-directed repair (HDR) Cas 9: is A protein enzyme that cuts both strands of DNA at sites specified by an RNA guide. Compare between tumor suppressor genes & oncogenes Tumor Suppressor Genes Protooncogenes-Oncogenes Inhibit Proliferation Stimulate Proliferation Stimulate Cell death Inhibit Cell death examples P53 examples growth factors, c Erb RB Retinoblastoma (first discovered in retinoblastoma) Compare between proto-oncogene & oncogene Proto-oncogene Oncogene codes for proteins that help to regulate the Altered protooncogene due to mutations or cell growth and differentiation. increased expression. A proto-oncogene is a normal gene List the protein controlling cell cycle check points: 1. 1-Cyclin-cyclin dependent kinases 2. 2-Retinoblastoma protein (Rb) 3. 3-Elongation factor 2F (E2F) 4. 4-Cell cycle accelerators 5. 5-Cell cycle suppressors List tumor suppressor genes. p53 Retinoblastoma protein CDKIs 29 List the functions of Tumor suppressor gene. 1-Regulate Cell Growth: Control the cell cycle and prevent uncontrolled cell division. 2-DNA Repair: Fix damaged DNA to maintain genomic stability 3-Induce Apoptosis: Trigger cell death in damaged or abnormal cells. 4-Prevent Metastasis: Stop cancer cells from spreading to other tissues 5-Inhibit Angiogenesis: Block the formation of blood vessels needed for tumor growth. What are the molecular pathways altered with cancer? I. unbalanced cell growth. II. Tumor Angiogenesis (It must grow its own blood vessels). III. Metastasis (Cells move away from the original (1ry) site and invade other organs) Describe the mechanisms of proto-oncogene activation 1. Point mutation (change in protein structure producing hyperactive protein) 2. Gene Amplification (increase protein expression) 3. Chromosomal translocation (Example Burkitt’s lymphoma) Placing two genes from two different chromosomes -----Forming fused gene-----Produce abnormal protein---- --Stimulate cell growth 30  4. Insertional mutagenesis Integration of viral DNA into the host genome---- can activate proto-oncogenes ----(e.g., insertion of HPV DNA leading to cervical cancer). List the functions of P53. 1-DNA Damage Response: p53 regulates genes involved in DNA repair. 2-Cell Cycle Regulation: p53 induces cell cycle arrest, allowing time for DNA repair before cell division 3-Apoptosis Induction: to prevent the proliferation of potentially cancerous cells. Pathway of P53 If DNA damage: - p53 makes the choice between cell cycle arrest and apoptosis Discuss the effect of mutation or deletion of P53: 1. Mutations in the P53 gene are found in more than half of all human cancers. 2. These mutations often result in a loss of function. 3. Loss of p53 function removes critical cell cycle checkpoints and apoptosis pathways 4. allowing cells with damaged DNA to proliferate, leading to tumorigenesis 31