Biochemistry Notes

Biochemistry Biochemistry Dr.Mayur Sayta Contect details Mob.Number- 9714941350 90169413...

Biochemistry Biochemistry Dr.Mayur Sayta Contect details Mob.Number- 9714941350 9016941350 SF-1,2 Samarth tower E Near- Waghodiya Chokdi Baroda- 390025 Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 1  Biochemistry ENDOPLASMIC RETICULUM (ER) It is a network of interconnecting membranes  It is continuous from outer nuclear envelope to outer plasma membrane.  It is reticular arrangement  It is prominent in cells which are actively synthesizing proteins  Proteins, glycoproteins and lipoproteins are synthesized in the ER.  Detoxification of various drugs is an important function of ER.  ER is classified into two varieties.  Rough and Smooth  ER looks rough due to ribosomes. It is related with protein synthesis.  Smooth ER is related with Lipid synthesis. MITOCHONDRIA Mitochondria is spherical, oval or rod-like bodies.  Mitochondria have two membranes. Inner membrane and outer membrane.  The inner membrane having folds also known as cristae  The inner membrane contains the enzymes of electron transport chain  The fluid contains the enzymes of citric acid cycle, urea cycle, etc..  Mitochondria are the powerhouse of the cell.  In mitochondria different metabolites are oxidized and energy is stored in the form of ATP.  Erythrocytes do not contain mitochondria. Cytochrome P-450 system present in mitochondrial inner membrane is involved in Steroid synthesis.  Mitochondria also contain specific DNA.  Inner membranes proteins are made by own DNA.  Other majority of proteins, especially of outer membrane are synthesized from cellular DNA. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 2  Biochemistry PLASMA MEMBRANE The plasma membrane separates the cell from the external environment.  It is semi permeable.  So entry and exit of compounds are regulated.  The membrane is metabolically very active. Structure  The structure of the membrane was described as a fluid mosaic model by Singer and Nicolson.  Membranes are mainly made up of lipids, proteins and small amount of carbohydrates.  The contents of these compounds vary according to the nature of the membrane. Membrane Carbohydrates  Carbohydrates are present as glycoproteins and glycolipids. Membrane Lipid  Phospholipids are the most common lipids.  The phospholipids are arranged in bilayers.  Polar head arranged towards outside and hydrophobic parts towards inside.  Cell membranes also contain cholesterol.  The lipid bilayer shows free movement of its components, so it is also called as fluid in nature. Membrane Proteins  The peripheral proteins are situated on the surfaces of the bilayer.  The integral membrane proteins are situated inside the bilayer.  The integral membrane proteins which are arranged in whole bilayer called transmembrane proteins.  The transmembrane proteins can work as receptors. 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For more information log on to www.mayursayta.com Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 4  Biochemistry CLASSIFICATION OF AMINO ACIDS 1. Based on Structure 1-A. Aliphatic amino acids a. Mono amino mono carboxylic acids: Simple amino acids: Glycine, Alanine Branched chain amino acids: Valine, Leucine, Isoleucine Hydroxy amino acids: Serine, Threonine Sulphur containing amino acids: Cysteine, Methionine Amino acids with amide group: Asparagine, Glutamine b. Mono amino dicarboxylic acids: Aspartic acid, Glutamic acid Di basic mono carboxylic acids: Lysine, Arginine 1-B. Aromatic amino acids: Phenylalanine, Tyrosine 1-C. Heterocyclic amino acids: Tryptophan, Histidine 1-D. Imino acid: Proline 1-E. Derived amino acids: 1-E-i. Derived amino acids found in proteins: After the synthesis of proteins, some of the amino acids are modified,  hydroxy proline and hydroxy lysine are important components of collagen.  Gamma carboxylation of glutamic acid residues of proteins is important for clotting process.  In ribosomal proteins and in histones, amino acids are extensively methylated and acetylated. 1-E-ii. Derived amino acids not seen in proteins (Non-protein amino acids):  Some derived amino acids are seen free in cells, e.g. Ornithine , Citrulline, Homocysteine. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 5  Biochemistry  These are produced during the metabolism of amino acids.  Thyroxine may be considered as derived from tyrosine. 1-E-iii. Non-alpha amino acids:  Gamma amino butyric acid (GABA) is derived from glutamic acid.  Beta alanine, where amino group is in beta position, is a constituent of pantothenic acid (vitamin) and co-enzyme A. 2. CLASSIFICATION BASED ON SIDE CHAIN 2-A. Amino acids having nonpolar side chains:  These include Alanine, Valine, Leucine, Isoleucine,  Aromatic amino acids Methionine, Proline, Phenylalanine and Tryptophan.  These groups are hydrophobic (water repellant) and lipophilic. Therefore, the parts of proteins made up of these amino acids will be hydrophobic in nature. 2-B. Amino acids having uncharged or nonionic polar side chains:  Glycine, Serine, Threonine, Cysteine, Tyrosine, Glutamine and Asparagine belong to this group.  These amino acids are hydrophilic in nature. (Tyrosine and Cysteine may show hydrophobic character when present in the interior of the protein). 2-C. Amino acids having charged or ionic polar side chains (hydrophilic): C-a. Acidic amino acids: They have a negative charge on the R group: Aspartic acid and Glutamic acid (Tyrosine is mildly acidic). C-b. Basic amino acids: They have a positive charge on the R group: Lysine, Arginine and Histidine 3. CLASSIFICATION BASED ON METABOLISM 3-A. Purely Ketogenic Leucine is purely ketogenic because it is converted to ketone bodies 3-B. Ketogenic and Glucogenic  Lysine, Isoleucine, Phenylalanine, Tyrosine and Tryptophan are partially ketogenic and partially glucogenic.  How ever in humans lysine is predominantly ketogenic. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 6  Biochemistry  During metabolism, part of the carbon skeleton of these amino acids will enter the ketogenic pathway and the other part to glucogenic pathway. 3-C. Purely Glucogenic  All the remaining 14 amino acids are purely glucogenic as they enter only into the glucogenic pathway 4. CLASSIFICATION BASED ON NUTRITIONAL REQUIREMENTS 4-A. Essential or Indispensable The amino acids may further be classified according to their essentiality for growth.  Thus Isoleucine, Leucine, Threonine, Lysine, Methionine, Phenylalanine, Tryptophan, and Valine are essential amino acids.  Their carbon skeleton cannot be synthesized by human beings and so preformed amino acids are to be taken in food for normal growth. 4-B. Partially essential or Semi-essential  Histidine and arginine are semi-indispensable amino acids.  Growing children require them in food. But they are not essential for the adult individual. 4-C. Non-essential or Dispensable  The remaining 10 amino acids are non-essential, because their carbon skeleton can be synthesized Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 7  Biochemistry Que. STRUCTURE OF PROTEINS (Organisation of Proteins) Proteins have different levels of structural organization. 1. Primary structure of protein means the order of amino acids in the polypeptide chain. 2. Secondary structure means the relationship of amino acids, close to each other. 3. Tertiary structure means interrelationship of various regions of a single polypeptide chain. 4. Quaternary structure results when the protein is made up of more than one polypeptide chains joined together. 1. Primary Structure Primary structure of protein means the order of amino acids in the polypeptide chain.  Each polypeptide chain has a unique amino acid sequence.  That sequence is decided by the genes.  The primary structure is maintained by the covalent peptide bonds.  The peptide bond is a partial double bond. Numbering of Amino Acids in Proteins  In a polypeptide chain, at one end there will be one free amino group.  This end is called the amino terminal (N-terminal) and the amino acid is named as the first amino acid.  At the other end of chain there is a free carboxyl group.  It is known as carboxy terminal (C-terminal) and the amino acid is named as the last amino acid.  Other amino and carboxyl groups are involved in peptide bond formation. Primary Structure Determines Biological Activity of protein  Higher levels of structural organization are dependent on the primary structure.  Even a single amino acid change can affect the function of protein.  For example, in HbA (normal hemoglobin) the 6th amino acid in the beta chain is glutamic acid; it is changed to valine in HbS (sickle cell anemia). Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 8  Biochemistry 2. Secondary Structure of Proteins Secondary structure means the relationship of amino acids, close to each other. Secondary level of protein structure is maintained by noncovalent forces. Described below  Hydrogen bond  Ionic bonds  Hydrophobic bonds  Van der Waals forces Alpha helix The alpha-helix is the most common and stable conformation for a polypeptide chain.  The alpha helix is a spiral structure  The structure is stabilized by hydrogen bonds between NH and C=O groups of the amino acids.  Each turn is formed by 3.6 residues. And each turn is 5.4 Å.  The distance between each amino acid residue is 1.5 Å. The alpha-helix is generally right handed.  Proline and hydroxy proline will not allow the formation of alpha-helix. Beta-pleated sheet  In beta sheet, The distance between adjacent amino acids is 3.5Å.  It is stabilized by hydrogen bonds between NH and C=O groups of neighboring polypeptide segments. Parallel beta sheet  If Adjacent strands in a sheet run in the same direction with regard to the amino and carboxy terminal ends of the polypeptide chain known as parallel Anti parallel beta sheet  If Adjacent strands in a sheet can run in the opposite direction known as anti parallel beta sheet. Beta-pleated sheet is the major structural organization in proteins like silk Fibroin  Beta sheets may be formed in many proteins by the U-turn folding of the chain. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 9  Biochemistry Tertiary Structure  Tertiary structure means interrelationship of various regions of a single polypeptide chain.  The tertiary structure denotes three dimensional structure of the whole protein.  The tertiary structure is maintained by noncovalent interactions such as hydrophobic bonds, electrostatic bonds and van der Waals forces.  Domains are generated in tertiary structure. Quaternary Structure  Quaternary structure results when the protein is made up of more than one polypeptide chains joined together.  The quaternary structure is maintained by hydrogen bonds, electrostatic bonds, hydrophobic bonds and van der Waals forces.  Depending on the number of polypeptide chain, the protein may be termed as monomer (1 chain), dimer (2 chains), tetramer (4 chains).  For example, 2 alpha-chains and 2 beta-chains form the Hemoglobin molecule. Que. Denaturation of Proteins  Change in protein structure is known as denaturation. It occurs due to o Mild heating, o Treating with acid and alkali o X-ray, ultraviolet rays  There will be alterations in secondary, tertiary and quaternary structures of protein molecules.  Primary structure is not changed during denaturation Due to denaturation  Solubility is decreased  Precipitability of the protein is increased.  Biological activity of protein is lost.  Denatured proteins have more sites for enzyme action so easily digested. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 10  Biochemistry  Cooking leads to denaturation of proteins so cooked foods are easily digested.  Most of the time It is irreversible process.  Denatured proteins are sometimes re-natured when the physical agent is removed. Ex. Hemoglobin denaturation can be reversed CLASSIFICATION OF PROTEINS The following classifications are given… Classification based on functions  Catalytic proteins, e.g. enzymes  Structural proteins, e.g. collagen  Contractile proteins, e.g. myosin, actin.  Transport proteins, e.g. hemoglobin, myoglobin, albumin  Regulatory proteins or hormones, e.g. ACTH, insulin, growth hormone  Genetic proteins, e.g. histones  Protective proteins, e.g. immunoglobulins, interferons Classification based on Composition 1. Simple Proteins  According to definition, they contain only amino acids. Albumins, Globulins 2. Conjugated Proteins  They are combinations of protein with a non-protein part.  Conjugated proteins are as follows:  Glycoproteins- containing glycogen  Lipoproteins- containing Lipid 3. Derived Proteins  They are degradation products of native proteins.  Progressive hydrolysis of protein results in smaller and smaller chains: Protein → peptones → peptides → amino acids. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 11  Biochemistry Classification Based on the Shape 1. Globular Proteins  They are spherical or oval in shape. 2. Fibrous Proteins  The molecules are elongated or needle shaped. Classification Based on Nutritional Value 1. Nutritionally Rich Proteins  They contain all the essential amino acids in the required proportion. 2. Poor Proteins  They lack in many essential amino acids Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 12  Biochemistry Transamination Transamination is the process in which amino group is transferred from amino acid to keto acid and forming a new amino acid. amino acid 1 + keto acid 2  amino acid 2 + keto acid 1  Example, amino group is interchanged between alanine and glutamic acid.  In almost all cases, the amino group is accepted by alpha ketoglutaric acid so that glutamic acid is formed.  The enzymes take part in the reaction known as amino transferases.  These enzymes have pyridoxal phosphate as prosthetic group.  The reaction is reversible. Biological Significance of Transamination 1. First step of degradation of aminoacid  In this first step of degradation of aminoacid, amino group is removed and given to the alpha ketoglutarate to form glutamate.  So transamination becomes first step for the degradation of aminoacid. 2. Synthesis of nonessential amino acids  By transamination, nonessential amino acids can be synthesized.  It is done by transferring amino group to keto acids.  For example, when pyruvate gets amino group by transamination, it is converted into alanine. Those amino acids, which cannot be synthesized in this manner, are therefore essential They should be taken into food. 3. Interconversion of amino acids  If amino acid no.1 is high and no.2 is low  The amino group from no.1 may be transferred to a keto acid to give amino acid no. 2 to equalize the quantity of both aminoacids.  This is called equalization of quantities of nonessential amino acids. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 13  Biochemistry 4. Clinical Significance of Transamination  Aspartate amino transferase (AST) and Alanine amino transferase (ALT) are induced by glucocorticoids which favor gluconeogenesis.  AST and ALT are markers of liver injury. Trans-deamination It is the process by which free amino group is released from amino acid. After transamination, oxidative deamination occurs.  Transamination takes place in the cytoplasm of all the cells of the body.  Amino group is transported to liver in the form of glutamic acid  It is finally deaminated in the mitochondria of hepatocytes and release free Ammonia. Oxidative Deamination of Glutamate  Only liver mitochondria contain glutamate dehydrogenase (GDH)  It deaminates glutamate to form alpha keto glutarate and ammonia.  So, all amino acids are first transaminated to glutamate, which is then deaminated (transdeamination).  glutamate dehydrogenase reaction is the final reaction which removes the amino group. The hydrolysis of glutamine also produces NH3.  This occurs mainly in the kidney  In kidney NH4+ production occurs which is helpful for acid base regulation. Minor Pathways of Deamination  Ammonia may be formed in the body through minor reactions like oxidation of monoamines by MAO (monoamine oxidase) Nonoxidative Deaminations  Dehydratases act on amino acids to remove ammonia from the following amino acids: Serine, Threonine  Desulfhydrase removes ammonia group from Cysteine Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 14  Biochemistry UREA CYCLE The cycle is known as Krebs–Henseleit urea cycle. As ornithine is the first member of the reaction, it is also called as Ornithine cycle. The two nitrogen atoms of urea are derived from two different sources, one from ammonia and the other directly from aspartic acid.  Formation of Carbamoyl Phosphate One molecule of ammonia condenses with CO2 in the presence of two molecules of ATP to form Carbamoyl phosphate.  Further cycle is described below… Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 15  Biochemistry Compartmentalization  First two enzymes are in the mitochondrial matrix.  Others are in cytosol. Energetics of Urea Cycle The overall reaction may be summarized as: NH3 + CO2 + Aspartate  Urea + fumarate  During these reactions, 2 ATPs are used in the 1st reaction.  Another ATP is converted to AMP + PPi in the 3rd step, which is equivalent to 2 ATPs.  The urea cycle use 4 phosphate bonds.  However, fumarate formed in the 4th step may be converted to malate.  Malate when oxidised to oxaloacetate produces 1 NADH equivalent to 2.5 ATP. So net energy expenditure is only 1.5 high energy phosphates. The urea cycle and TCA cycle are interlinked, and so, it is called as "urea bicycle". Regulation of the Urea Cycle Coarse Regulation  It depends on protein content of diet.  During starvation, the activity of urea cycle enzymes is elevated to meet the increased rate of protein catabolism. Fine Regulation  The major regulatory step is by CPS-I Disorders of Urea Cycle  Deficiency of any of the urea cycle enzymes would result in hyperammonemia.  Further results in encephalopathy and respiratory alkalosis.  Clinical symptoms include vomiting, irritability, and severe mental retardation. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 16  Biochemistry ONE-CARBON METABOLISM One-carbon (1C) groups play a pivotal role in donating carbon atoms for synthesis of different types of compounds.  The different one-carbon groups of the ‘one-carbon pool' of the body are: o Formyl group o Formimino group o Methenyl group o Hydroxymethyl group o Methylene group o Methyl group  The one-carbon groups, except methyl group, are carried by tetrahydrofolic acid (THFA).  THFA is produced from folic acid. Generation of One-Carbon Groups It is by amino acids. 1. Serine to glycine is the primary contributor for methylene THFA. 2. Glycine cleavage system also produces methylene groups 3. Histidine contributes to N5-formimino THFA. 4. Tryptophan donates formyl-THFA 5. Choline is donor of hydroxyl methyl groups. Interconversion of One-Carbon Groups The different one-carbon groups are interconvertible. From methyl-THFA, the B12 co-enzyme accepts the methyl group to form methyl cobalamin.  Methyl cobalamin transfers the methyl group to homocysteine to form methionine.  This is the reaction in human metabolism, where B12 acts as a coenzyme  In B12 deficiency, deficiency of folic acid is also observed  This is because, the transfer of methyl group from methyl-THFA does not occur.  THFA is not regenerated; this is called folate trap. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 17  Biochemistry The one carbon units are used for synthesis of the following compounds: 1. C2 of purine 2. Formylation of methionyl tRNA 3. C8 of purine 4. Glycine (Fig. 15.1) 5. Serine 6. Choline (Fig.15.12) 7. Deoxy TMP (Fig. 14.17). 8. Transmethylation reactions including creatine, choline and epinephrine synthesis 9. Excreted as carbon dioxide Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 18  Biochemistry PHENYL KETONURIA (PKU) Deficiency of phenyl alanine hydroxylase is the cause for this disease.  Frequency of PKU was considered to be 1 in 10,000 births  Incidence of PKU in India is 1 in 25,000 births. Biochemical Abnormalities  Phenylalanine could not be converted to tyrosine.  So phenylalanine accumulates.  Phenylalanine level in blood is elevated.  So alternate minor pathways are opened  Phenyl ketone (phenyl pyruvate), phenyl lactate and phenyl acetate are formed and excreted in urine. Clinical Manifestations  Child is mentally retarded with an IQ of 50.  Agitation, hyperactivity, tremors and convulsions are often manifested. This may be because phenylalanine interferes with neurotransmitter synthesis.  The child often has hypopigmentation due to decreased level of tyrosine.  Phenyl lactic acid in sweat may lead to mousy body odor. Laboratory Diagnosis A. Blood phenylalanine: Normal level is 1 mg/dl.  In PKU, the level is >20 mg/dl. B. Guthrie test is positive in PKU C. Ferric chloride test: Urine of the patient contains phenyl ketones.  This could be detected by adding a drop of ferric chloride to the urine.  Blue-green color is a positive test. D. DNA probes are now available to diagnose the defects in phenylalanine hydroxylase deficiency. Treatment  Early detection is very important.  The treatment is to give a diet containing low phenyl alanine. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 19  Biochemistry ALKAPTONURIA Deficiency of homogentisate oxidase is the cause for this disease. Biochemical Defect  Alkaptonuria is an autosomal recessive condition.  Incidence- 1 in 250,000 births.  The metabolic defect is the deficiency of homogentisate oxidase  This results in excretion of homogentisic acid in urine.  The homogentisic acid is oxidized to benzoquinone acetate  It is then produce black colored alkaptone bodies. Clinical Manifestations  Person has almost normal life. The only abnormality is the blackening of urine on standing.  By the 3rd or 4th decade of life, patient may develop ochronosis (deposition of alkaptone bodies in intervertebral discs, cartilages of nose, pinna of ear).  Black pigments are deposited over the connective tissues including joint cavities to produce arthritis. Diagnosis of Alkaptonuria  Urine becomes black on standing when it becomes alkaline. Blackening is accelerated on exposure to sunlight and oxygen. The urine when kept in a test tube will start to blacken from the top layer.  Ferric chloride test will be positive for urine.  Benedict's test is strongly positive. Treatment  No specific treatment is required.  But minimal protein intake with less phenylalanine is recommended. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 20  Biochemistry ALBINISM The Greek word, albino means white.  Albinism is an autosomal recessive disease  Incidence- 1 in 20,000 population Biochemical Defect  Tyrosinase is completely absent, leading to defective synthesis of melanin. Clinical Manifestations  The ocular fundus is hypopigmented and iris may be grey or red.  There will be associated photophobia and decreased visual acuity.  The skin has low pigmentation, and so skin is sensitive to UV rays.  The skin may show presence of melanomas.  Hair is also white. Albinism may be produced by the following causes: a. Melanocyte deficiency b. Failure of melanocytes to form melanosomes. c. Due to tyrosinase deficiency, melanin is not produced in the melanosomes. d. Failure of melanosomes to form melanin owing to substrate deficiency. e. Failure of melanosomes to store melanin or to transport melanin to keratinocytes. f. Excessive destruction of functional melanosomes. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 21  Biochemistry Maple Syrup Urine Disease (MSUD) It is also called branched chain ketonuria.  The incidence is 1 per 1 lakh births  The name originates from the characteristic smell of urine (similar to burnt sugar or maple sugar) due to excretion of branched chain keto acids. Biochemical defect  Deficient decarboxylation of branched chain keto acids (BKA)  valine, leucine and isoleucine metabolism becomes abnormal.  Branched chain keto acids are produced.  So branched chain keto acids and valine, leucine and isoleucine.are excreted in urine. Clinical findings  Disease starts in the first week of life.  It is characterized by convulsions, severe mental retardation, vomiting, acidosis, coma and death within the first year of life. Laboratory findings  Urine contains branched chain keto acids, valine, leucine and isoleucine.  Rothera's test is positive  Diagnosis depends on enzyme analysis in cells. Treatment  Giving a diet low in branched chain amino acids.  Mild variant is called intermittent branched chain ketonuria.  This will respond to high doses of thiamine. This is because the decarboxylation of the BKA requires thiamine. Liver transplantation has been successfully tried in some cases of MSUD. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 22  Biochemistry Que. STRUCTURE OF HEMOGLOBIN Hemoglobin is made up of Heme+Globin.  Hb is globular in shape.  The adult Hb (HbA) has 2 alpha chains and 2 beta chains.  Hb F (fetal Hb) is made up of 2 alpha and 2 gamma chains.  Hb A2 has 2 alpha and 2 delta chains. Normal adult blood contains 97% HbA, about 2% HbA2 and about 1% HbF.  Alpha chain gene is on chromosome 16 while the beta, gamma and delta chains are on chromosome 11.  Each alpha chain has 141 amino acids. The beta, gamma and delta chains have 146 amino acids.  There are 36 histidine residues in Hb molecule; these are important in buffering action. The alpha and beta subunits are connected by relatively weak non-covalent bonds like vander Waals forces, hydrogen bonds and electrostatic forces. Normal level of Hemoglobin (Hb) in blood in males is 14-16 g/dl and in females, 13-15 g/dl. Attachment of Heme with Globin Chain  There are 4 heme residues per Hb molecule,one for each subunit in Hb.  The 4 heme groups account for about 4% of the whole mass of Hb.  The iron atom of heme occupies the central position of the porphyrin ring.  The reduced state is called ferrous (Fe++) and the oxidized state is ferric (Fe+++).  The ferrous iron has 6 valencies and ferric has 5 valencies.  In hemoglobin, iron remains in the ferrous state. Iron carries oxygen  The iron is linked to the pyrrole nitrogen by 4 coordinate valency bonds  Fifth valency of iron to nitrogen of the histidine.  the 6th valency of iron binds the O2. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 23  Biochemistry BIOSYNTHESIS OF HEME  Heme is synthesized in the normoblasts, but not in the matured erythrocytes. The pathway is partly cytoplasmic and partly mitochondrial. Step 1: ALA synthesis  The synthesis starts with the condensation of succinyl CoA and glycine in the presence of pyridoxal phosphate to form delta amino levulinic acid (ALA)  Hence anemia may be manifested in pyridoxal deficiency.  The enzyme ALA synthase is located in the mitochondria and  It is the rate-limiting enzyme of the pathway. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 24  Biochemistry Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 25  Biochemistry Porphyria Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 26  Biochemistry ELECTROPHORESIS electrophoresis refers to the movement of charged particles through an electrolyte when subjected to an electric field. Normal Patterns and Interpretations In agar gel electrophoresis, normal serum is separated into 5 bands. Their relative concentrations are given below:  Albumin : 55-65%  Alpha-1-globulin : 2- 4%  Alpha-2-globulin : 6-12%  Beta-globulin : 8-12%  Gamma-globulin : 12-22%  Albumin has the maximum and gamma globulin has the minimum mobility in the electrical field.  Gamma globulins contain the antibodies.  Most of the alpha-1 fraction is made up of alpha-1-antitrypsin.  Alpha-2 band is mainly made up by alpha-2-macroglobulin.  Beta fraction contains low density lipoproteins. Abnormal Patterns in Clinical Diseases Various abnormalities can be identified in the electrophoretic pattern.  Chronic infections: The gamma globulins are increased  Multiple myeloma: In para-proteinemias, a sharp spike is noted and is termed as M-band.  This is due to monoclonal origin of immunoglobulins in multiple myeloma.  Primary immune deficiency: The gamma globulin fraction is reduced.  Nephrotic syndrome: All proteins except very big molecules are lost through urine, and so alpha-2 fraction (containing macroglobulin) will be very prominent.  Cirrhosis of liver: Albumin synthesis by liver is decreased, with a compensatory excess synthesis of globulins by reticulo-endothelial system. So albumin band will be thin.  Chronic lymphatic leukemia- gamma globulin fraction is reduced.  Alpha-1-antitrypsin deficiency: The alpha-1 band is thin or even missing. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 27  Biochemistry ALBUMIN Albumin constitutes the major part of plasma proteins.  It is synthesized by hepatocytes  Normal level of Albumin is 3.5–5 g/dl. Functions of Albumin 1. Colloid osmotic pressure of plasma  proteins cannot easily escape out of blood vessels, and therefore, proteins exert the ‘effective osmotic pressure’. It is about 25 mm Hg  The maintenance of blood volume is dependent on this effective osmotic pressure.  If protein concentration in serum is reduced then return of water into blood vessels is diminished, leading to accumulation of water in tissues. This is called edema. 2. Transport Function  Albumin is the carrier of various hydrophobic substances in the blood.  Bilirubin, Drugs , Hormones: steroid hormones, thyroxine, Metals can be transported by albumin. 3. Buffering action  Albumin has buffering capacity. 4. Nutritional function  All tissue cells can take up albumin  It is then broken down to amino acid level. Clinical Applications Therapeutic use  Human albumin is therapeutically useful to treat burns, hemorrhage and shock. Edema Hypo-albuminemia will result in tissue edema  Malnutrition, where albumin synthesis is depressed (generalized edema)  Nephrotic syndrome, where albumin is lost through urine (facial edema)  Cirrhosis of liver (mainly ascites), where albumin synthesis is less and it escapes into ascitic fluid.  Chronic congestive cardiac failure: Venous congestion will cause increased hydrostatic pressure and decreased return of water into capillaries and so pitting edema of feet may result. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 28  Biochemistry Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 29  Biochemistry Enzymes Enzymes are biocatalysts.  They increase the rate of biochemical reaction.  They are not consumed or changed in that reaction.  At the end of biochemical reaction same enzyme is free to catalyse the next reaction. Structure of enzymes-  They are protein or polypeptide in nature except for a few.  They are heat labile. Que. What is endoenzymes?  Most enzymes function within the cell so called as endoenzymes. E.g. enzymes of glycolysis, tca cycle, fatty acid synthesis and other metabolic processes Que. What is exoenzymes?  Some enzymes function outside the cell called as exoenzymes. E.g. enzymes of digesion. Que. What is proenzymes?  Some enzymes are excist as an inactive precursor form called as proenzymes e.g trypsin is secreted by pancreas as inactive trypsinogen  it is secreted inside gi tract and than gets activated by enterokinase and converted to trypsin.  Their names have prefix “pro” or suffix “ogen” like chymotrypsinogen, trypsinogen, pepsinogen, prophospholipase. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 30  Biochemistry Que. CLASSIFICATION OF ENZYMES Class 1. Oxidoreductases: This class of enzymes related with oxidation or reduction process.  e.g. Lactate dehydrogenase  Glucose-6-phosphate dehydrogenase Class 2: Transferases This class of enzymes transfers a group from the substrate to another substrate.  Example- Hexose + ATP → Hexose-6-phosphate + ADP.  The name of enzyme is Hexokinase. Class 3: Hydrolases This class of enzymes can hydrolyse different bonds by adding water and then breaking the bond.  Example- Acetyl choline + H2O --------→ Choline + acetate  The enzyme is Acetyl choline esterase.  All digestive enzymes are hydrolases. Class 4: Lyases This class of enzymes can break bonds by mechanisms other than hydrolysis.  For example- Fructose-1,6-bisphosphate -------→ Glyceraldehyde-3- phosphate +dihydroxy acetone phosphate (DHAP).  The enzyme is Aldolase Class 5: Isomerases This class of enzymes can produce isomers of substrates.  Examples- Glyceraldehyde-3-phosphate -----→ Di-hydroxy-acetone- phosphate  Enzyme is Triose phosphate isomerase. Class 6: Ligases This class of enzymes link two substrates together.  For Example- Dna Ligase Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 31  Biochemistry Que. Coenzyme  Enzyme contain chemical group other than amino acids.  Enzymes associated with non-protein chemical component which is required for catalytic activity is known as co-enzyme. Holoenzyme- apoenzyme + cofactor/coenzyme  The complete enzyme is known as holoenzyme which is made up of protein portion known as apoenzyme and co-enzyme.  apoenzyme is inactive without coenzyme Coenzyme can be divided into two broad groups  Organic cofactors, such as flavin or heme  Inorganic cofactors, such as the metal ions Mg2+, Cu+, Mn2+, or iron-sulfur clusters. Organic cofactors are sometimes further divided into co-substrate and prosthetic groups. co-substrate  It is related temporary with active site of enzyme.  In a group transfer reaction co-substrate acts either a donor or acceptor of the group.  For example- pyridoxal phosphate [a part of transamination enzyme] helps in transfer of amino group from an aminoacid to a ketoacid.  It works as temporary carrier of the transferred aminoacid. Prosthetic group-  Prosthetic group is bound permanently to the active site of the enzyme.  "prosthetic group" emphasizes the nature of the binding of a cofactor to a protein (tight or covalent)  For example biotin is the prosthetic group for carboxylase enzyme.  It is the integral component for enzyme. It is attached strongly with apoenzyme component. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 32  Biochemistry Classification of co-enzyme It can be classified according to group whose transfer they facilitate For transfer group other than hydrogen-  Examples  pyridoxal phosphate for amino group transfer  ATP for phosphate group transfer  Biotin for carbon dioxide transfer. For transfer of hydrogen  Examples  e.g. NAD+ , NADP+, FAD NAD+ o It is a co-enzyme synthesised from nicotinamide a member of vit-B Coplex. o Hydrogen is accepted by this complex to form NADH. Metallo-enzymes  These are enzymes which require certain metal ions for their activity  Zink is required for carbonic anhydrase  Magnesium and Manganese are required for hexokinase, enolase  Iron is required for cytochrome oxidase, catalase  Calcium is required for lipase QUE-Factors affecting Enzyme Activity  The various factors which affect the enzyme activity are explained below Enzyme concentration  Rate of reaction or velocity is directly proportional to the enzyme concentration.  Velocity of reaction is increased with the increase in enzyme concentration. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 33  Biochemistry Substrate concentration-  As substrate concentration is increased the enzyme which bind with the substrate also increasing and velocity of reaction increase.  When all the enzymes are bind with substrate, the maximum velocity is obtained.  If we add further substrates, velocity is not increased because all the enzymes are utilised Products concentration  In a reversible reaction when product concentration is increased the reaction rate is slowed down. Temperature  As temperature increases, initially the rate of reaction will increase, because of increased Kinetic Energy.  The temperature at which the maximum rate of reaction occurs is called the enzyme's Optimum Temperature.  This is different for different enzymes.  Most enzymes in the human body have an Optimum Temperature of around 37.0 °C.  As temperature increased further, enzymes shapes will be changed  Enzymes will be Denatured.  This will decrease the rate of reaction pH - Acidity and Basicity  Different enzymes have different Optimum pH values.  This is the pH value at which the bonds within them are influenced by H+ and OH- Ions in such a way that the shape of their Active Site is the most Complementary to the shape of their Substrate. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 34  Biochemistry  At the Optimum pH, the rate of reaction is at an optimum.  Any change in pH above or below the Optimum will quickly cause decrease in the rate of reaction.  Usually enzymes have optimum ph between 6 and 8.  Some exceptions are pepsin [optimum ph 1-2] alkaline phosphatase [optimum ph 9-10] and acid phosphatase [optimum ph 4-5] presence of activator  Activator activate the inactive enzyme and influence enzyme activity presence of inhibitors  Competitive, non-competitive inhibitors modify enzymetic activity. Que-Enzyme inhibition Catalytic activity of the enzymes can be inhibited by certain chemical compound.  In enzyme inhibitor combines with enzyme and prevents normal enzyme substrate interaction  This determine the rate of the reaction  Two main types of inhibitors are recognised, reversible and irreversible. Reversible inhibitor  Reversible inhibitor binds to enzyme through non-covalent bond.  And activity of enzyme is restored fully when the inhibitor is removed. There are following types of reversible inhibitors competitive inhibitor  Also known as substrate analogue inhibitor  There is a competition between inhibitor and normal substrate for catalytic binding site of the enzyme.  This is because both the inhibitor and the substrate have similar structural configuration.  Therefore enzyme cannot differentiate and both can bind to enzyme. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 35  Biochemistry  BUT Only ES can form product.  E+S+I  ES+EI  ESE+P where E= enzyme, S= substrate, I= inhibitor, P= Product  Competitive inhibition can be reversed by increasing substrate concentration. non-competitive inhibitor  In this type of inhibition substrate and inhibitor are structurally different than each other so there is no competition.  Inhibitor binds to enzyme at a site other than the substrate binding site. When it binds the shape of enzyme is changed so that its catalytic activity is reduced or lost.  Substrate is able to bind with enzyme, but the enzyme cannot catalyse the reaction when inhibitor is bound.  It can form EI or ESI complex and inhibit enzymetic activity Irreversible inhibitor  Irreversible inhibitor binds covalently with an enzyme to form a stable complex, so that the enzyme is permanently inactivated Suicide inhibition-  These compounds are unreactive until they bind to active site of specific enzyme.  Enzyme binds with them and converts them into reactive compound which bind irreversibly to enzyme and inhibition occurs.  Ex. Allopurinol is a suicide inhibitor of xanthine oxidase Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 36  Biochemistry Que. Allosteric modulation  Enzymes posses a site in addition to substarte binding site is known as allosteric site.  And that enzyme is called as allosteric enzyme.  Binding of modulator at the allosteric site affects conformation of substrate binding site.  Such regulators are called as allosteric modulator Allosteric activation If modulator increase substrate binding known as positive allosteric modulator and process is allosteric activation  ex. AMP,ADP are positive modulator for enzyme phosphofructokinase I ,  AMP for pyruvate dehydrogenase,  acetyl coA for pyruvate carboxylase Allosteric inhibition  If modulator prevent the conformational change required for binding of the substrate to enzyme known as negative allosteric modulator  and process is allosteric inhibition.  Ex.ATP, citrate are negative modulator for enzyme phosphofructokinase I  ATP for pyruvate dehydrogenase Feedback allosteric Inhibition  It is a common mechanism of regulation in multi enzyme system.  Product of pathway inhibits the enzyme. o Ex. Delta aminolevulinic acid synthase is an allosteric enzyme which is inhibited by end product heme. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 37  Biochemistry ISO-ENZYMES They are physically distinct forms of the same enzyme.  Multiple molecular forms of an enzyme are described as iso-enzymes or isozymes.  Different molecular forms of the same enzyme synthesized from various tissues are called iso-enzymes. Hence study of iso-enzymes is very useful to understand diseases of different organs. Example  LDH 1 is increased than LDH 2 in myocardial infaction  CPK MB is increased in myocardial infaction  If the subunits are all the same, the protein is a homomultimer.  If the subunits are different, protein is said to be a heteromultimer. Identification of Iso-enzymes  In Agar gel or polyacrylamide gel electrophoresis, the iso-enzymes have different mobility.  LDH, CK and ALP iso-enzymes can be separated by electrophoresis. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 38  Biochemistry Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 39  Biochemistry Que. STRUCTURE OF DNA Deoxyribonucleic acid (DNA) is composed of four deoxy ribonucleotides, i.e. deoxyadenylate (A), deoxyguanylate (G), deoxycytidylate (C), and thymidylate (T).  These units are combined through phospho diester bonds  The nucleotide is formed by a combination of base + sugar + phosphoric acid. Polarity of DNA molecule  In the case of DNA, the base sequence is written from the 5' end to the 3' end.  This is called the polarity of the DNA chain. Watson-Crick Model of DNA Structure The salient features of Watson-Crick model of DNA are given below 1. Right handed double helix  DNA consists of two polydeoxy ribonucleotide chains  They are twisted around one another in a right handed double helix similar to a spiral stair case. 2. The base pairing rule  Always the two strands are complementary to each other.  So, the adenine pair with thymine of the opposite strand  Guanine will pair with cytosine. The base pairing (A with T; G with C) is called Chargaff's rule, which states that the number of purines is equal to the number of pyrimidines. 3. Hydrogen bonding  The DNA strands are held together mainly by hydrogen bonds between the purine and pyrimidine bases.  There are two hydrogen bonds between A and T while there are three hydrogen bonds between C and G. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 40  Biochemistry  The GC bond is therefore stronger than the AT bond. 4. Antiparallel  The two strands in a DNA molecule run antiparallel  Which means that one strand runs in the 5' to 3' direction, while the other is in the 3' to 5' direction. This is similar to a road divided into two, each half carrying traffic in the opposite direction. 5. Other features In the DNA, each strand acts as a template for the synthesis of the opposite strand during replication process.  The spiral has a distance of 34 A per turn.  Within a single turn, 10 base pairs are seen.  Thus, adjacent bases are separated by 3.4 A  The diameter or width of the helix is 20 A. Que. Transfer RNA (tRNA) They transfer amino acids from cytoplasm to the ribosome. So, the name is transfer RNA.  Each molecule is only 73-93 nucleotides in length Structure of tRNA Molecule  The transfer RNAs show extensive internal base pairing and clover leaf like structure Acceptor Arm is at the 3' End  It carries the amino acid  This area has 7 base pairs.  The end sequence is CCA-3'. Anticodon Arm of tRNA  At the opposite side of the acceptor arm is the anticodon arm  It recognizes the triplet nucleotide Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 41  Biochemistry codon present in mRNA.  The specificity of tRNA resides in the anticodon site. It has base sequences complementary to that of mRNA codon.  For example, if the mRNA has a codon with the sequence UUU  the anticodon sequence of the tRNA will be AAA, by which it base pairs with mRNA codon. DHU Arm of tRNA  The D arm or DHU region is so named due to the presence of a dihydro uridine in that area  The DHU arm serves as the recognition site for the enzyme which adds the amino acid. Pseudouridine Arm of tRNA  The opposite side of D arm is called pseudouridine arm, as it contains a pseudouridine.  It is involved in binding tRNA to ribosomes. Variable arm tRNA molecules possess a variable arm  a short extra arm 3 to 5 base pairs long and they belong to Class 1 tRNA  The tRNA molecules belonging to Class 2, have a long extra arm, 13 to 21 base pairs in length. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 42  Biochemistry Que. REPLICATION OF DNA Process of copying the DNA is known as DNA replication.  In the daughter cell, one strand is derived from the mother cell; while the other strand is newly synthesized.  This is called semiconservative type of DNA replication.  Each strand serves as a template over which a new strand is synthesized  The base pairing rule is always maintained.  The new strand is joined to the old strand by hydrogen bonds between base pairs (A with T and G with C) The whole process can be studied under the following steps:  Site of replication is identified by origin recognition complex (ORC).  DNA helicase unwinds the DNA. o Helicases move on both directions. It is separating the strands for the replication. o This forms a replication bubble with two replication forks  Topo- isomerases – Removal of supercoil is done by topo- isomerases. o It breaks one strand of DNA, Remove coiling and join it.  The protein A binds at specific sites of origin, and opens the dna strand.  Single stranded DNA binding proteins (SSB) stabilize the complex. DNA is prevented from recoiling by SSBs.  An RNA primer, about 100-200 nucleotides long, is synthesized by the RNA primase at the starting of DNA rreplication. DNA Polymerase (DNAP)  This enzyme synthesises a new strand of DNA, by adding Neucleotides. Bacterial DNA polymerases  DNA polymerase III is the main replication enzyme in bacteria.  DNA polymerase I is a repair enzyme. It has both 3' to 5' and 5' to 3' exonuclease activities. Mammalian DNA Polymerases (DNAP) In mammalian cells (eukaryotic), there are 5 DNAPs, named as α, β, γ, δ, ε  alpha is the major enzyme responsible for chromosome replication. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 43  Biochemistry  beta DNAP is a repair enzyme.  DNAP gamma is concerned with mitochondrial DNA synthesis.  Delta enzyme is used for both leading and lagging strand synthesis.  Epsilon is used for leading strand synthesis. Elongation of DNA Strand The DNA polymerase adds nucleotide complementary to template strand.  Formation of the new strand of DNA is taking place from 5' to 3' direction.  The strand continuously Synthesized is known as the “leading strand”  The strand which is not continuously synthesized is known as the “lagging strand”  DNA portion attached to primer RNA called as Okazaki fragments.  Then the RNA primer is removed by DNAP.  New DNA portion is synthesized at this part.  Previously formed DNA and This DNA are sealed by the DNA ligase.  Further winding of DNA is done. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 44  Biochemistry Que. TRANSCRIPTION Transcription includes initiation, elongation, and termination steps followed by post-transcriptional processing. Signals for Initiation of Transcription A. Promoters  There are certain specific areas on the DNA that act as starting signals for initiation process.  The RNAP attaches at the promoter site on the template DNA strand. B. TATA Box in Prokaryotes  In the case of bacteria, about 35 bp before the transcription start site, there is a sequence of 5'-TGTTGACA-3'  About 10 bp before, there is another sequence 5'-TATAAT-3'. This one is referred to as TATA box. Initiation of Transcription  The DNA helix partially unwinds, and the RNA Polymerase binds with the promoter site on DNA  This is called pre-initiation complex (PIC). Elongation Process of Transcription  The RNAP moves along the DNA template.  New nucleotides are added one by one, according to the base pairing rule  Thus A in DNA is transcribed to U in mRNA; T to A; G to C and C to G.  The synthesis of mRNA is from 5' to 3' end.  RNAP has no nuclease activity  So, there is no proof reading.  Mistake rate in mRNA transcription is more than DNA replication. 5. Termination of Transcription  The specific signals are recognized by a termination protein. The Rho factor  Rho factor attaches to the DNA, the RNAP cannot move further and consequently newly formed mRNA is released  Rho independent termination can also occur.. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 45  Biochemistry Post-transcriptional Processing In mammalian system, newly synthesized RNA [primary transcript] undergoes modifications to become the mature mRNA. These modifications are:  Endonuclease cleavage  Poly-A tailing  Methylation  Removal of introns  Splicing of exons (connect together). These processings occur mainly in the nucleoplasm.  In bacteria, mRNA is not changed.  Post-transcriptional processing is also done for tRNA and rRNA. Poly-A Tailing at 3' End  At The 3' terminus poly-A tail may be 20 to 250 nucleotides long is added.  This tail protects mRNA from attack by exonuclease. Capping at 5' End  Eukaryotic mRNAs are all 'capped' at the 5' terminus by 7-methyl guanosine triphosphate.  The cap is useful in recognition of mRNA by the translating machinery. Methylations  These are mainly done in the cytoplasm. Removal of Introns  The primary transcripts are very long  Large portions of primary RNA are removed.  The primary transcript contains coding regions (exons) joined with noncoding regions (introns).  These intron sequences are removed  Exons are combined together to form the mature mRNA molecule. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 46  Biochemistry Que. Genetic Code A triplet sequence of nucleotides on the mRNA is genetic code.  Since there are four different bases, they can generate 64 different codons. Important features of genetic code. 1. Nonoverlapping  The codes are consecutive.  The codes are read one after another in a continuous manner. 2. Nonpunctuated  There is no punctuation between the codons. 3. Degenerate  61 codes stand for the 20 amino acids.  So one amino acid has more than one codon.  For example, glycine has 4 codons.This is called degeneracy of the code. 4. Specificity  Though the codons are degenerate, they are Specific; or without any doubtful meaning.  That is, one codon stands only for one amino acid. 5. Universal  The codons are the same for the same amino acid in all species; the same for "Elephant and E.coli".  The genetic code has been highly preserved during evolution. 6. Terminator Codons  There are three codons which do not code for any particular amino acids.  They are “nonsense codons”, terminator codons.  They put "full stop" to the protein synthesis.  These three codons are UAA, UAG, and UGA. 7. Initiator Codon  In most of the cases, AUG acts as the initiator codon.  AUG also acts as the codon for methionine.  In a few proteins, GUG may be the initiator codon. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 47  Biochemistry Que. TRANSLATION PROCESS Translation is a process of protein synthesis from RNA.  The enzymes aminoacyl tRNA synthetases activate the amino acids.  In eukaryotes, the first amino acid incorporated is methionine (AUG codon).  The first AUG triplet is identified by the ribosome as the start codon.  tRNA carrying methionine and 40S ribosomal subunit are Joined to form pre-initiation complex.  This complex binds with mRNA to form initiation complex  Initiation complex now binds with 60S ribosomal unit to form the full assembly of 80S ribosome. P and A Sites of Ribosomal Assembly  The whole ribosome contains two sites for tRNA molecules.  The “P” site or peptidyl site carries the peptidyl-tRNA. It carries the growing peptide chain.  The “A” site or aminoacyl site carries the new incoming tRNA with the amino acid to be added next. Elongation Process of Translation Elongation has 3 steps  Binding of aminoacyl tRNA to the A site  Peptide bond formation occurs between two aminoacids.  Translocation of the ribosome on the mRNA.  Again new tRNA comes with aminoacid and same steps repeated. Termination Process of Translation  After successive addition of amino acids, ribosome reaches the terminator codon sequence (UAA, UAG or UGA) on the mRNA.  And further synthesis stops.  Finally 80S ribosome dissociates into its component units of 60S and 40S Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 48  Biochemistry Post-translational Processing A. Proteolytic cleavage  Modification of polypeptides by partial proteolysis. e.g. conversion of pro-insulin to insulin B. Modifications of amino acids  Gamma carboxylation of glutamic acid residues of prothrombin, under the influence of vitamin K.  Hydroxylation of proline and lysine in collagen with the help of vitamin C.  Phosphorylation of hydroxyl groups of serine, threonine or tyrosine by kinases, e.g. glycogen phosphorylase. C. Subunit aggregation  Examples are immunoglobulin, hemoglobin and maturation of collagen. Failure of post-translational modification affects the normal function of many protein. Que. MUTATIONS A mutation is defined as a change in nucleotide sequence of DNA. Classification of Mutations A point mutation is defined as change in a single nucleotide. This may be subclassified as (a) substitution, (b) deletion, and (c) insertion Substitution  Replacement of a purine by another purine ( A to G or G to A) or pyrimidine by pyrimidine (T to C or C to T) is called Transition mutation.  If a purine is changed to a pyrimidine (e.g. A to C) or a pyrimidine to a purine (e.g. T to G), it is called a transversion. The point mutation present in DNA is transcribed and translated, so that the defective gene produces an abnormal protein. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 49  Biochemistry Deletion Deletions may be subclassified into–  Large gene deletions  Deletion of a codon  Deletion of a single base Insertion Insertions or additions or expansions are subclassified into–  Single base additions, leading to frameshift effect.  Trinucleotide expansions.  Duplications. Effects of Mutations Silent Mutation  Codon is changed but amino acid is not changed.  Then the mutation is silent and has no effect Mis-sense but Acceptable Mutation  A change in amino acid may be produced in the protein; but with no functional problem.  It is acceptable Mutation. Mis-sense; Partially Acceptable Mutation  In this type, the amino acid is changed and it affects the functional properties of the protein.  HbS or sicklecell hemoglobin Mis-sense; Unacceptable Mutation  The single amino acid substitution alters the properties of the protein to such an extent that it becomes nonfunctional and the condition is incompatible with normal life.  For example, HbM [met-hemoglobinemia.] Nonsense; Terminator Codon Mutation  A codon may be changed to a termination codon (UAA or UAG).  This leads to premature termination of the protein,  So, functional activity may be destroyed. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 50  Biochemistry Frameshift Mutation  This is due to addition or deletion of bases.  From that point onwards, the reading frame shifts.  In this deletion or addition of base changes all the triplet codons thereafter.  Therefore, a useless protein or no protein is produced. Que. Lac Operon Operon is a unit of gene expression. it includes, structural genes, control elements, regulator inhibitor gene, promoter and operator areas.  In the bacterial cell, the Z gene encodes beta galactosidase The enzyme which hydrolyzes lactose to galactose and glucose.  The Y gene is responsible for production of a permease which transports lactose and galactose into the cell.  The A gene codes for thiogalactoside transacetylase. Since Z, Y and A code for the structure of the proteins, they are called structural genes. These 3 genes are present as contiguous segments of DNA The transcription of these genes start from a common promoter (P), located close to the Z gene. The RNA polymerase binds to the promoter and transcribes these 3 structural genes as a single mRNA. Transcription is Normally Repressed  Transcription of the structural gene is under the control of another regulator or the "i" (inhibitor) gene.  The lac repressor has strong affinity to the operator site..  The operator site is between the promoter and structural genes Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 51  Biochemistry When RNAP identifies the promoter sequence and moves towards the structural genes, it is stopped by repressor molecule. This is like the action of a zip. If a thread is placed across its way, the zip cannot move further. Similarly, when repressor is attached to the operator, RNAP cannot move further. So structural genes are not transcribed.  Thus, when lactose is not available, the lactose utilizing enzymes are not synthesized Derepression of Lac Operon  When lactose is introduced into the medium, lactose binds to the repressor protein.  Repressor-lactose complex is inactive, which does not bind to the operator region.  So there is no repressor molecule at the operator site.  Now, RNAP can transcribe the structural genes, which are then translated Thus lactose switches the genes "on". Lactose induces the synthesis of lactose utilizing enzymes.  Hence lactose is an inducer of these genes Such regulation, where several proteins are regulated as one unit, is termed as coordinate gene regulation. Que. Recombinant DNA technology The basic principles 1. Generation of DNA fragments and selection of the desired piece of DNA (e.g. human gene). 2. Insertion of the selected DNA into a cloning vector (e.g. plasmid) to create a recombinant DNA or chimeric DNA 3. Introduction of the recombinant vecrors into host cells (e.g. bacteria). 4. Multiplication and selection of clones containing the recombinant DNA. 5. Expression of the gene to produce the desired product. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 52  Biochemistry Recombinant DNA technology with special reference to the following aspects is described  Molecular tools of genetic engineering.  Host cells- The factories of cloning.  Vectors- The cloning vehicles.  Methods of gene transfer  Gene cloning strategies. Molecular tools of genetic engineering Restriction endonucleases - DNA cutting enzymes  Restriction endonucleaseas are one of the most important groups of enzymes for the manipulation of DNA.  These are enzymes that can cut DNA at specific sites.  They were first discovered in E.coli  The cut DNA fragments by restriction endonucleases may have mostly sticky ends or blunt ends  DNA fragments with sticky ends are particularly useful for recombinant DNA DNA ligases-DNA joining enzymes  The cut DNA fragments are covalently joined together by DNA ligases. These enzymes were originally isolated from viruses.  They also occur in E.coli and eukaryotic cells.  DNA ligases actively participate in cellular DNA repair process. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 53  Biochemistry Purines synthesized from Que. Salvage Pathway Through this pathway recycling of purines occur.  Purines which are formed by degradation of nucleotides are recycled by this pathway.  Nucleosides and deoxy-nucleosides can also be salvaged. PRPP is the starting material in this pathway; it is also a substrate for de novo synthesis.  Hence these two pathways are closely interrelated. The free purines are salvaged by two different enzymes  Adenine phospho ribosyl transferase (APRTase)  Hypoxanthine guanine phosphoribosyl transferase (HGPRTase). The pathway is of special importance in tissues like RBCs and brain where the de novo pathway is not operating.  The salvage pathway economizes intracellular energy expenditure.  Absence of enzymes of salvage pathway produces specific clinical syndromes. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 54  Biochemistry Salvage pathway is summarized below: APRTase Adenine + PRPP ---------------- AMP + PPi HGPRTase Guanine + PRPP ---------------- GMP + PPi HGPRTase Hypoxanthine + PRPP --------------- IMP + PPi Disorders of Purine Metabolism The most common abnormality is an elevation of uric acid level in blood, referred to as hyperuricemia. It is defined as serum uric acid concentration exceeding 7 mg/dl in male and 6 mg/dl in female. Que. GOUT It is due to accumulation of urate crystals in the synovial fluid resulting in inflammation leading to acute arthritis.  uric acid is filtered into joint and binds with sodium to form sodium urate crystals  They are mainly deposited in distal joints of the Body. Gout may be either primary or secondary. Primary Gout Causes of primary gout are: 1. Over activity of phosphoribosyl amido transferase  This would lead to overproduction of purine nucleotides. 2. Increased activity of PRPP synthetase  Leads to increased production of PRPP. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 55  Biochemistry 3. Deficiency of enzymes of salvage pathway  HGPRT deficiency 4. Glucose-6-phosphatase deficiency  This condition is known as Von Gierke's disease (glycogen storage disease).  When this enzyme is deficient, glucose-6-phosphate cannot be converted to glucose.  So more glucose is channelled into the pentose-phosphate shunt pathway  resulting in increased availability of ribose- 5-phosphate.  This would lead to increased formation of PRPP. 5. Glutathione reductase increased activity Secondary Gout When the gout is associated with other Disease known as Secondary Gout.  Renal failure.  Leukemia or other cancer Clinical Findings of Gout  Gouty attack increased by increased intake of alcohol.  It mainly affects first metatarsophalangeal joint (big toe), but other joints also affected.  The joints are extremely painful.  Synovial fluid will show urate crystals. In chronic cases  In chronic gout, the deposition of urate crystals in renal medulla occurs which progresses to Renal stone Treatment in Gout  Restrict alcohol.  Probenecid - Increase renal excretion of uric acid.  Allopurinol – it is a competitive inhibitor of xanthine oxidase so decreasing formation of uric acid.  Aspirin - For reducing Pain Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 56  Biochemistry Lesch-Nyhan Syndrome It is an X-linked inherited disorder of purine metabolism. Incidence is 1:10,000 males. There is deficiency of HGPRTase.  So, the rate of salvage pathway is decreased resulting in accumulation of PRPP and decreased level of inhibitory purine nucleotides. The disease is characterized by  self mutilation,  mental retardation,  excessive uric acid  Renal stone Gout develops in later life. The neurological manifestations suggest that the brain is dependent on the salvage pathway for the requirements of IMP and GMP. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 57  Biochemistry VITAMIN A Chemistry  Vitamin A is fat soluble. beta-carotene is present in plant tissues. Three different compounds with vitamin A activity are  retinol (vitamin A alcohol),  retinal (vitamin A aldehyde) and  retinoic acid (vitamin A acid) Daily Requirement of Vitamin A The recommended daily allowance (RDA) for  Children = 400-600 microg/day.  Men = 750-1000 microg/day  Women = 750 microg/day, Pregnancy = 1000 microg/day Dietary Sources of Vitamin A  Animal sources include milk, butter, cream, cheese, egg yolk and liver.  Fish liver oils  Carrot ,Papaya, mango,  green leafy vegetables Absorption of Vitamin A  Beta carotene is broken down to form retinal.  The retinal is converted to retinol  The absorption of retinol occurs along with other fats  Within the mucosal cell, the retinol is esterified with fatty acids  It is transported to liver by chylomicrons  VitaminA is stored in the form of retinol palmitate. Transport from Liver to Tissues  The vitamin A from liver is transported to peripheral tissues by the retinol binding protein or RBP Uptake by Tissues  The retinol-RBP complex binds to specific receptors on the retina, skin, gonads and other tissues. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 58  Biochemistry  Vitamin A transpoted inside cell and binds to cellular retinoic acid binding protein (CRBP)  That complex binds to hormone responsive elements (HRE) of DNA.  Thus, genes are activated  They form different proteins helpful for growth and differentiation of cell Biochemical Role of Vitamin A Wald's Visual Cycle Generation of Nerve Impulse  Rhodopsin plays main in vision.  Rhodopsin is made up of the protein opsin and 11-cis-retinal.  When light falls on the retina, the 11-cisretinal converted to all-trans- retinal.  And generation of nerve impulse occurs. Regeneration of 11-cis-retinal  After dissociation, all-trans-retinal is isomerized to 11-cisretinal in the retina itself in the dark by the enzyme retinal isomerase. Alternatively, all-trans-retinal is transported to liver  It is reduced to all-trans-retinol by alcohol dehydrogenase (ADH),  The all-trans-retinol is isomerized to 11-cis-retinol  Then it is oxidised to 11-cisretinal.  This is transported to retina. This completes the Wald's visual cycle  Rods are for Vision in Dim Light  Cones are for Color Vision Other Biochemical Functions of Vitamin A  Retinoic acid has a role in the regulation of gene expression and differentiation of tissues.  Retinol is necessary for the reproductive system.  Anti-oxidant property: o The anticancer activity has been noticed due to antioxidant activity Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 59  Biochemistry  Beta carotenes may be useful in preventing heart attacks  Vitamin A is necessary for the maintenance of normal epithelium and skin. Deficiency manifestations of Vitamin A Night Blindness  Visual acuity is decreased in dim light. Xerophthalmia  The conjunctiva becomes dry, thick. Bitot's Spots  These are seen as triangular spots attached to the conjunctiva.  This is due to increased thickness of conjunctiva Keratomalacia  There is degeneration of corneal epithelium  Bacterial infection leads to corneal ulceration, and total blindness. Skin and Mucous Membrane Lesions  The alterations in skin may cause increased occurrence of generalized infections.  Decreased development of mucous membrane. Other General Manifestations  In vitamin A deficiency, growth retardation especially failure of skeletal growth is noticed. Causes for Vitamin A Deficiency  Decreased intake  Liver Damage which results in decreased RBP. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 60  Biochemistry VITAMIN D (CHOLECALCIFEROL) Formation of Vitamin D  Vitamin D is derived from 7-dehydrocholesterol by the action of ultraviolet radiations.  It is converted in to cholecalciferol  The cholecalciferol is first transported to liver, where hydroxylation at 25th position occurs, to form 25-hydroxy cholecalciferol (25-HCC).  In the kidney, it is further hydroxylated at the 1st position.  Thus 1,25-dihydroxy cholecalciferol (DHCC) is generated. Since it contains three hydroxyl groups at 1, 3 and 25 positions, it is also called Calcitriol (Fig. 33.9).  The calcitriol is the active form of vitamin D. Requirement of Vitamin D (RDA)  Children = 10 microgram (400 IU)/day  Adults = 5 microgram (200 IU)/day  Pregnancy, lactation = 10 microgram/day Sources of Vitamin D  Exposure to sunlight  fish liver oil, fish and egg yolk , Milk Biochemical Effects of Vitamin D Vitamin D and Absorption of Calcium Calcitriol increase the absorption of calcium and phosphorus from the intestine. Calcitriol acts like a steroid hormone.  It enters the target cell and binds to a cytoplasmic receptor.  The hormone-receptor complex interacts with DNA  And transcription of specific genes that code for specific protein.  Due to the increased availability of calcium binding protein, the absorption of calcium is increased. Effect of Vitamin D in Bone  Vitamin D increasing Mineralization of the bone by increasing the activity of osteoblasts Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 61  Biochemistry Effect of Vitamin D in Renal Tubules  Calcitriol increases the reabsorption of calcium and phosphorus by renal tubules Regulation of Calcitriol  The level of calcitriol is maintained by the feedback control.  The rate of production is modulated by serum levels of calcium, phosphorus, PTH and calcitriol itself. Deficiency of Vitamin D The deficiency diseases are rickets in children and osteomalacia in adults.  Hence vitamin D is known as antirachitic vitamin. Causes for Vitamin D Deficiency Deficiency of vitamin D can occur in people who are not exposed to sunlight properly Clinical Features of Rickets  Rickets is seen in children.  There is insufficient mineralization of bone.  Bones become soft.The bone growth is reduced.  Weight bearing bones are bent  The clinical manifestations include bow legs, knock-knee and pigeon chest. Clinical Features of Osteomalacia  The bones are softened due to insufficient mineralization.  Patients are more prone to get fractures. Renal rickets:  In kidney diseases, even if vitamin D is available, calcitriol is not synthesized.  These cases can be treated by giving calcitriol. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 62  Biochemistry Deficiency of Thiamine (Beriberi) Deficiency of thiamine leads to beriberi. It is a word, meaning "weakness".  The early symptoms are anorexia, dyspepsia, heaviness and weakness.  Subjects feel weak and get easily exhausted. Wet beriberi: Here cardiovascular system is more affected.  Edema of legs, face, trunk and serous cavities are the main features.  Palpitation, breathlessness are also seen.  Death occurs due to heart failure. Dry beriberi In this condition CNS is more affected.  Walking becomes difficult.  Peripheral neuritis with sensory disturbance leads to complete paralysis. Infantile beriberi It occurs in infants born to mothers suffering from thiamine deficiency.  Restlessness and sleeplessness are seen in infant. Wernicke-Korsakoff syndrome:  It is also called as cerebral beriberi.  Clinical features are like encephalopathy ( cerebellar ataxia)  Psychosis is seen only when the nutritional status is severely affected. Polyneuritis: It is common in chronic alcoholics.  Alcohol metabolism needs large doses of thiamine.  Alcohol inhibits intestinal absorption of thiamine, leading to thiamine deficiency.  It may cause problem of conversion of pyruvate to acetyl CoA.  This results in increased plasma concentration of pyruvate and lactate, leading to lactic acidosis. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 63  Biochemistry Niacin Deficiency Pellagra Deficiency of niacin leads to the clinical condition called pellagra. Pellagra is an Italian word, meaning "rough skin". Pellagra is caused by the deficiency of Tryptophan as well as Niacin. Dermatitis  Redness occurs on skin of feet, ankles and face.  Increased pigmentation around the neck.  The dermatitis is increased by exposure to sunlight. Diarrhea  The diarrhea may be mild or severe. It may contain blood and mucus.  This may lead to weight loss.  Nausea and vomiting may also be present. Dementia  Irritability, inability to concentrate and poor memory  Ataxia and spasticity are also seen. Niacin is Synthesized from Tryptophan About 60 mg of tryptophan is equivalent to 1 mg of niacin. Causes for Niacin Deficiency Dietary deficiency of Tryptophan  Pellagra is seen among people whose staple diet is maize  In maize, niacin is present; but it is in a bound form, and is unavailable. Deficient synthesis:  Kynureninase, an important enzyme in the pathway of tryptophan  It is dependent on pyridoxine  Conversion of tryptophan to niacin is not possible in pyridoxal deficiency. Deficient absorption from intestine  Intestinal disorders. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 64  Biochemistry ASCORBIC ACID (VITAMIN C) Chemistry of Vitamin C It is water soluble. The structural formula of ascorbic acid closely resembles that of carbohydrates  Chemical name- Ascorbic acid Biosynthesis of Ascorbic Acid in Animals  Most animals and plants can synthesize ascorbic acid from glucose.  Man, higher animals cannot synthesize ascorbic acid because of deficiency of gulono lactone oxidase enzyme. Dietary Sources of Vitamin C  amla, guava, lime, lemon and green leafy vegetables. Requirement of Vitamin C  RDA is 75 mg/day  During pregnancy, lactation requirement is 100 mg/ day. Metabolism of Ascorbic Acid  Ascorbic acid is readily absorbed from gastrointestinal tract.  The vitamin is excreted in urine. Biochemical Functions of Vitamin C Reversible oxidation-reduction It can change between ascorbic acid and dehydroascorbic acid. Hydroxylation of proline and lysine  Ascorbic acid is necessary for the post-translational hydroxylation of proline and lysine residues  Hydroxyproline and hydroxylysine are necessary for the formation of cross links in the collagen  It gives strength to the fibres.  This process is necessary for the normal production of collagen and intercellular cement substance of capillaries. Tryptophan metabolism  Ascorbic acid is necessary for the hydroxylation reaction during tryptophan metabolism Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 65  Biochemistry Tyrosine metabolism  Vitamin C helps in the oxidation reaction during metabolism of tyrosine Iron metabolism  ascorbic acid convert ferric iron to ferrous iron.  So, it increase the iron absorption from the intestine Hemoglobin metabolism  It is useful for re-conversion of met-hemoglobin to hemoglobin. Folic acid metabolism  Ascorbic acid is helping the enzyme folate reductase to reduce folic acid to tetrahydrofolic acid Steroid synthesis  vitamin C has role in steroid hormone synthesis.  Vitamin C helps in the synthesis of bile acids from cholesterol. Phagocytosis  Ascorbic acid stimulates phagocytic action of leukocytes  and helps in the formation of antibodies. Anti-oxidant property  As an anti-oxidant it may prevent cancer formation. Cataract  Regular intake of ascorbic acid reduces the risk of cataract formation. Deficiency Manifestations of Vitamin C Scurvy Deficiency of vitamin C results in scurvy. Hemorrhagic tendency  In ascorbic acid deficiency, collagen is abnormal and the intercellular cement substance is brittle  So capillaries are fragile, leading to increase chances of bleeding. Internal hemorrhage Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 66  Biochemistry  In severe cases, hemorrhage may occur in the conjunctiva and retina.  Internal bleeding may be seen as epistaxis, hematuria or melena. Oral cavity  the gum becomes painful, swollen and spongy Wound healing may be delayed. Bones  bone is weak and fractures easily. Anemia In vitamin C deficiency anemia is seen due to reduced absorption of iron and abnormal hemoglobin. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 67  Biochemistry BIOLOGICAL OXIDATION The transfer of electrons from the reduced coenzymes through the respiratory chain to oxygen is known as biological oxidation.  Energy released during this process is trapped as ATP. Electron Transport Chain The electron flow occurs through successive dehydrogenase enzymes together known as electron transport chain (ETC).  The electrons flow from electronegative potential (-0.32) to electropositive potential (+ 0.82). ORGANIZATION OF ELECTRON TRANSPORT CHAIN All the components of electron transport chain (ETC) are located in the inner membrane of mitochondria. ETC Complex-I  It is also called NADH-CoQ reductase complex.  It contains a flavoprotein (Fp) consisting of FMN and an iron-sulphur protein (FeS)  NADH is the donor of electrons, FMN accepts them and gets reduced to FMNH2  The electrons from FMNH2 are then transferred to Fe-S.  The electrons are then transferred to coenzyme Q. Complex II or Succinate-Q-Reductase  The electrons from FADH2 enter the ETC at the level of coenzyme Q.  This step does not liberate enough energy to act as proton pump. Coenzyme Q  It accepts a pair of electrons from NADH or FADH2 through complex-I or complex II respectively  The two electrons than transferred to Complex III Complex III or Cytochrome Reductase  This is a cluster of iron-sulphur proteins, cytochrome b and cytochrome c1, both contain heme prosthetic group. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 68  Biochemistry Cytochrome C  It is a peripheral membrane protein containing one heme prosthetic group.  Cytochrome c collects electrons from Complex III and delivers them to Complex IV. Complex IV or Cytochrome Oxidase  It contains different proteins, including cytochrome a and cytochrome a3.  Four electrons are accepted from cytochrome c, and passed on to molecular oxygen. ATP SYNTHASE (COMPLEX V)  It is a protein assembly in the inner mitochondrial membrane. It is sometimes referred to as the 5th Complex  Proton pumping ATP synthase is a multisubunit transmembrane protein.  It has two functional units, named as F1 and Fo. Mechanism of ATP synthesis:  ATP synthesis (oxidative phosphorylation) occurs through the interaction of F1 and Fo. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 69  Biochemistry Regulation of ATP Synthesis  The availability of ADP regulates the process.  When ATP level is low and ADP level is high, oxidative phosphorylation proceeds at a rapid rate. P:O Ratio  The P:O ratio is defined as the number of inorganic phosphate molecules incorporated into ATP for every atom of oxygen consumed.  When a pair of electrons from NADH reduces an atom of oxygen (½ O2), 2.5 mol of ATP are formed  1.5 ATP from FADH2. Current Concept, Energetics of ATP Synthesis  The proton gradient thus created is maintained across the inner mitochondrial membrane till electrons are transferred to oxygen to form water.  The electrochemical potential of this gradient is used to synthesize ATP  When one NADH transfers its electrons to oxygen, 10 protons are pumped out and approximately 3 ATP.  Similarly the oxidation of 1 FADH2 is accompanied by the pumping of 6 protons and approximately 2 molecules of ATP.  However, Peter Hinkle recently proved that the actual energy production is less, because there is always leakage of protons.  one NADH may generate only 2.5 ATP; and one FADH2 may generate only 1.5 ATP.  1 glucose will generate only 32 ATPs. 1 Palmitate gives 106 molecules CHEMI-OSMOTIC THEORY The coupling of oxidation with phosphorylation is termed oxidative phosphorylation.  Peter Mitchell has given a theory to explain the oxidative phosphorylation.  The transport of protons from inside to outside of inner mitochondrial membrane results in generation of a proton gradient across the membrane. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 70  Biochemistry  Protons (H+ ions) accumulate outside the membrane, creating an electrochemical potential difference.  This proton motive force drives the synthesis of ATP by ATP synthase complex. PROTON PUMP AND ATP SYNTHESIS  The proton pumps (complexes I, III and IV) expel H+ from inside to outside of the inner membrane.  So, there is high H+ concentration outside the inner membrane.  This causes H+ to enter into mitochondria through the channels (Fo); this proton influx causes ATP synthesis by ATP synthase  The pH outside the mitochondrial inner membrane is 1.4 units lower than inside.  Further, outside is positive relative to the inside (+0.14 V).  The proton motive force (PMF) is 0.224 V corresponding to a free energy change of 5.2 kcal/mol of protons. Trapped in the form of ATP. Que. Inhibitors of ATP synthesis Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 71  Biochemistry Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 72  Biochemistry DISACCHARIDES When two monosaccharides are combined together by glycosidic linkage, a disaccharide is formed. The important disaccharides are 1. Sucrose 2. Maltose and isomaltose 3. Lactose. Sucrose It is the sweetening agent known as cane sugar. It is present in sugarcane and various fruits. Sucrose contains glucose and fructose. Sucrose is not a reducing sugar; and it will not form osazone.  This is because the linkage involves first carbon of glucose and second carbon of fructose  Free reducing groups are not available. So… When sucrose is hydrolysed, the products have reducing action.  A sugar solution which is originally nonreducin  but becomes reducing after hydrolysis, is identified as sucrose (specific sucrose test) Sucrose hydrolysis is inversion  Hydrolysis of sucrose (optical rotation +66.5°) will produce one molecule of glucose (+52.5°) and one molecule of fructose (–92°).  Therefore the products will change the dextrorotation to levorotation  the plane of rotation is inverted.  Equimolecular mixture of glucose and fructose thus formed is called invert sugar.  The enzyme producing hydrolysis of sucrose is called sucrase or invertase.  Honey contains invert sugar. Invert sugar is sweeter than sucrose. Lactose It is the sugar present in milk. It is a reducing disaccharide.  On hydrolysis lactose produce glucose and galactose. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 73  Biochemistry  Beta glycosidic linkage is present in lactose.  The anomeric carbon atom of beta-galactose is attached to the 4th hydroxyl group of glucose through beta-1,4 glycosidic linkage.  Lactose forms osazone which resemble "badminton ball" or "hedgehog" or flower of "touch-me-not" plant. Maltose Maltose contains two glucose residues.  There is alpha-1,4 linkage, i.e.1st carbon atom of one glucose is combined with 4th carbon of another glucose through alpha-glycosidic linkage.  It is a reducing disaccharide.  It forms petal shaped crystals of maltose-osazone. Isomaltose It contains 2 glucose units combined in alpha -1, 6 linkage.  Thus first carbon of one glucose residue is attached to the sixth carbon of another glucose through a glycosidic linkage.  It is a reducing sugar  Partial hydrolysis of glycogen and starch produces isomaltose. POLYSACCHARIDES These are polymerized products of many monosaccharide units. They may be 1. Homoglycans are composed of single kind of monosaccharides, e.g. starch, glycogen and cellulose. 2. Heteroglycans are composed of two or more different monosaccharides, e.g. hyaluronic acid, chondroitin sulphate. Homopolysaccharides / homoglycanes 1. Starch A. Structure of Starch  It is the reserve carbohydrate of plant kingdom. Sources: Potatoes, tapioca, cereals (rice, wheat) and other food grains. Starch is composed of amylose and amylopectin. Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 74  Biochemistry  When starch is treated with boiling water, 10-20% is solubilized; this part is called amylose.  Amylose is made up of glucose units with alpha-1,4 glycosidic linkages  The insoluble part absorbs water and forms paste like gel; this is called amylopectin.  Amylopectin is also made up of glucose units, but is highly branched  The branching points are made by alpha-1,6 linkage. B. Hydrolysis of Starch Starch is non reducing.  When starch is hydrolysed , It produce smaller and smaller fragments are produced.  Thus hydrolysis produces amylodextrin.  Further hydrolysis produces erythrodextrin ,Than achrodextrins  Further it produce maltose and then glucose. Digestion Salivary amylase and pancreatic amylase are alpha-amylases  They act on alpha- 1,4 glycosidic bonds to split starch into smaller units (dextrins), and finally to alpha-maltose. 2. Glycogen  It is the reserve carbohydrate in animals.  It is stored in liver and muscle.  Excess carbohydrates are deposited as glycogen.  Glycogen is composed of glucose units joined by alpha-1,4 links in the straight chains.  It also has alpha-1,6 glycosidic linkages at the branching points  Glycogen is more branched and more compact than amylopectin. 3. Cellulose It is the supporting tissues of plants.  Cellulose is the most abundant organic material in nature.  It is made up of glucose units combined with beta-1,4 linkages. It has a straight line structure, with no branching points.  Beta-1,4 bridges are hydrolysed by the enzyme cellobiase.  But this enzyme is absent in animal and human digestive system, and hence Sayta Medical Coaching Institute www.mayursayta.com M- 9714941350 Page 75  Biochemistry  cellulose cannot be digested.  Herbivorous animals have large caecum, which harbor bacteria.  These bacteria can hydrolyse cellulose,and the glucose produced is utilized by the animal. 4. Inulin It is a long chain homoglycan composed of D-fructose units with repeating beta- 1,2 linkages.  It is clinically used to find renal clearance value and glomerular filtration rate. 5. Dextrans  These are highly branched homopolymers of glucose units with 1-6, 1-4 and 1-3 linkages.  They are produced by microorganisms. Since they will not easily go out of vascular compartment,  They are used for intravenous infusion as plasma volume expander for treatment of hypovolemic shock 6. Chitin  It is present in exoskeletons of insects.  It is composed of units of N-acetylglucosamine with beta-1,4 glycosidic