AQ-112 - Fundamentals of Biochemistry PDF

PRINCIPLES OF BIOCHEMISTRY Unit 1: Introduction Chapter 1: Introduction to Biochemistry 1.1.1 Introduction to Biochemistry is a branch of science, which deals with the chemistry of life and living Biochemistry processes. The term biochemistry is defined as a science concerned with the...

PRINCIPLES OF BIOCHEMISTRY Unit 1: Introduction Chapter 1: Introduction to Biochemistry 1.1.1 Introduction to Biochemistry is a branch of science, which deals with the chemistry of life and living Biochemistry processes. The term biochemistry is defined as a science concerned with the chemical nature and the chemical behaviour of living matter. Biochemical studies include the type of chemical constituents of living matter, their transformation in biological system and energy changes associated with these transformations. Biochemistry links biology and chemistry by studying how complex chemical reactions and chemical structures give rise to life and life's processes. It incorporates everything in size between a molecule and a cell and all the interactions between them. It deals with the study of the structure and function of cellular components and the processes carried out both on and by organic macromolecules-especially proteins, carbohydrates, lipids, nucleic acids, and other biomolecules. 1.1.2 History of Biochemisry Important foundations were laid in many fields of biology during 17th and 18th centuries. The development of very crucial concepts, which include the cell theory by Schleiden and Schwann in 1833, Mendel’s study of inheritance in 1866 and Darwin’s theory of evolution were observed during 19th century. The real boon to biochemistry was given in 1828 when total synthesis of urea from ammonia and lead cyanate was successfully achieved by Wohler who thus demonstrated that organic compound can be synthesized from inorganic compound. During 1857, Louis Pasteur did a great deal of work on fermentation and pointed out categorically the importance of enzymes in this process. In 1897, Edward Buchanner extracted enzymes from yeast cells in crude form which could a sugar molecule to alcohol and thus enzyme research was initiated. Neuberg first introduced the term, ‘biochemistry’ during 1903. In 1905, Knoop deduced the β- Oxidation reactions for fatty acid degradation.In 1972, Jon Singer and Garth Nicolson proposed the fluid mosaic model of membrane structure. The early part of 20th century witnessed a sudden increase in knowledge related to chemical analysis, separation methods and instrumentation for biological studies (X-ray diffraction, electron microscope etc.) which led to the understanding in structure and function of important molecules such as proteins, enzymes, DNA and RNA. In 1926 James Sumner established the protein nature of enzymes. The first metabolic pathway, glycolysis, was elucidated by Embden and Meyerhof.in1933. Otto Warberg, Cori and Parnas also made important contributions relating to glycolytic pathway. Citric acid and urea cycles were established by Krebs during 1930-40. The central role of ATP in biological systems was described by Lipmann in 1940. The The double-helical model of DNA was established by Watson and Crick in 1953. DNA polymerase was discovered by Kornberg in 1956. Frederick Sanger’s contributions in sequencing of protein in 1953 and nucleic acid in 1977 were responsible for further developments in the fields of protein and nucleic acids. The development of recombinant DNA research by Snell in 1980 led to the growth in the field of genetic engineering. Thus there is progressive evolution of biology to, biochemistry to molecular biology, genetic engineering and biotechnology. The research still grows. 1.1.3 Structure and function of Cell The cell is the basic unit of life. All organisms are made up of cells (or in some cases, a single cell) and depend on cells to function normally. There are two major classifications of cells: the prokaryotes and eukaryotes. The prokaryotes (Greek “pro” -primitive; “karyon” – nucleus) lack a nucleus. e.g. bacteria. Eukaryotic cells (Greek “eu” - good; “karyon” - nucleus) have a membrane enclosed nucleus encapsulating their DNA. e. g. Cells of animals, plants and fungi. a. Prokaryotes: Prokaryotes are almost always single-celled, except when they exist in colonies. Reproduce by means of binary fission. 1|Page b. Eukaryotes: Eukaryotic cells range in size from 1 to 100 micrometers in diameter and have volume a thousand to a million times that of a typical prokaryotic cell. Cells are covered by a cell membrane and come in many different shapes. The contents of a cell are called the protoplasm. i. Protoplasm: It is differentiated in to plasma membrane (=plasmalemma or cell membrane), cytoplasm, nucleus and vacuoles. Cytoplasm is distinguishable in to cytoplasmic matrix and endoplasmic reticulm. Cytoplasmic matrix is also called hyaloplasm. It is a polyphasic colloidal system which exists in two states, sol and gel. The gel form usually occurs near the plasma membrane. This region is sometimes called ectoplast in contrast to sol region known as endoplast. Ectoplast is firmer. It is quite conspicuous on the free sides of the cells. In protozoans, ectoplast is prominent on all sides. Cytoplasmic matrix is generally in perpectual motion. The phenomenon is called cyclosis, cytoplasmic or protoplasmic streaming. In the cytoplasmic matrix are embedded a large number of cell organells or organised protoplasmic subunits having specific functions. ii. Organelle: An organelle is a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own membrane. Organelles are identified by microscopy, and can also be purified by cell fractionation. Eukaryotic cells contain several types of organelles, while prokaryotic cells contain a few organelles (ribosomes) and none that are bound by a membrane. There are also differences between the kinds of organelles found within different eukaryotic cell types. Plant cells for example, contain structures such as a cell wall and chloroplasts that are not found in animal cells. The parts of a plant cell and an animal cell, important organelles and their functions are described herewith. 1.1.3.1 Cell membrane The cell membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is semi-permeable to ions and organic molecules and controls the movement of substances in and out of cells. It consists of the phospholipid bilayer with embedded proteins, which are involved in a variety of cellular processes such as cell signalling, cell adhesion and ion conductivity. The plasma membrane also serves as the attachment surface for the extracellular glycocalyx and cell wall of plant cell and intracellular cytoskeleton. 2|Page 1.1.3.2 Cytoplasm The cytoplasm is the part of a cell that is enclosed within the cell membrane. In the cells of prokaryote organisms, which lack a nucleus, the contents of are contained within the cytoplasm. In the cells of eukaryote organisms the contents of the cell nucleus are separated from the cytoplasm, and are there called the nucleoplasm. The cytoplasm contains organelles, which are filled with liquid that is kept separate from the rest of the cytoplasm by biological membranes. It is within the cytoplasm that most cellular activities occur, such as many metabolic pathways including glycolysis, and processes such as cell division. The inner, granular mass is called the endoplasm and the outer, clear and glassy layer is called the ectoplasm. The part of the cytoplasm that is not held within organelles is called the cytosol. The cytosol is a complex mixture of cytoskeleton filaments, dissolved molecules, and water that fills much of the volume of a cell. 1.1.3.3 Nucleus The nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. DNA directs the protein biosynthesis inside the cell. The main structures making up the nucleus are the Nuclear membrane, a double membrane that encloses the entire organelle. Many nuclear pores are inserted in the nuclearmembrane, which facilitate and regulate the exchange of materials (proteins, tRNA, mRNA and rRNA) between the nucleus and the cytoplasm. Chromatin is the combination of DNA and proteins. It is found inside the nuclei of eukaryotic cells. Nucleolus is an organelle within the nucleus from where ribosomal RNA is produced. Some cells have more than one nucleus. 1.1.3.4 Endoplasmic reticulum (ER) Endoplasmic reticulum(ER) is a vast system of interconnected, membranous, infolded and convoluted sacks that are located in the cell's cytoplasm (the ER is continuous with the outer nuclear membrane). They are of two types: Rough endoplasmic reticulum and Smooth endoplasmic reticulum. i) Rough endoplasmic reticulum (rough ER): Rough ER is covered with ribosomes that give it a rough appearance. Rough endoplasmic reticulum bears the ribosomes during protein synthesis. The newly synthesized proteins are sequestered in sacs, called cisternae. The system then sends the proteins via small vesicles to the golgi apparatus. In the case of membrane proteins it inserts them into the membrane ii) Smooth endoplasmic reticulum (smooth ER): Smooth ER buds off from rough ER. The space within the ER is called the ER lumen. Smooth endoplasmic reticulum transports the proteins manufactured by the rough endoplasmic reticulum to other locations in the cell or outside the cell. This is achieved through a process called budding, wherein the small vesicles, which contain proteins, are detached from the smooth endoplasmic reticulum and are carried to other locations. This cell organelle also aids in converting glucose-6-phosphate to glucose, which is an important step in gluconeogenesis 3|Page 1.1.3.5 Mitochondrion Mitochondrion is spherical to rod-shaped organelle with a double membrane. The inner membrane is infolded many times, forming a series of projections (called cristae).The space between the two membranes is called “outer chamber” or “inter membrane space”. It is filled, with a watery fluid and is 40-70Ǻ in width. The space bounded by inner membrane is called the “inner chamber” or “inner membrane space”. The inner membrane space is filled with a matrix. It is rich in enzymes and contains dense granules, ribosomes, and mitochondrial DNA. The granule consists of insoluble inorganic salts and are believed to be the binding sites of bivalent ions like Mg2+ and Ca2+. The side of the inner membrane facing the matrix side is called M-side, while the side facing the outer chamber is called C-side. Two to six circular DNA molecules have been identified within mitochondria. They may be present free in the matrix or may be attached to the membrane. The enzymes of the Krebs cycle are located in the matrix.In general the outer membrane contains more proteins and phospholipids than the inner membrane. Phosphatidylcholie is the predominant lipid of the outer membrane.The innermembrane on the other hand contains most of the diphophatidylglycerol. On the inner layer of cristae are found stalked particles or repeating triparticle units which are sometimes called respiratory assemblies. Each stalked particles or repeatng triparticle units which are sometimes consists of abase, a stalk spherical head. The spherical soluble coupling particle called F1 particle which is actually the enzyme ATPase, oxidative phosphorylation takes place on stalked particles. Mitochondria convert the energy stored in glucose into ATP (adenosine triphosphate) for the cell and hence are the power house of the cell. 1.1.3.6 Chloroplasts Chloroplasts are the green plastids. They are organelles found in plant cells and other eukaryotic organisms that conduct photosynthesis. They are usually disk- shaped and about 5-8 µm in diameter and 2-4 µm thick. A typical plant cell has 20-40 of them. Chloroplasts are green because they contain chlorophylls, the pigments that harvest the light used in photosynthesis. They capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis. The dark reaction of photosynthesis takes place in stroma. The material within the chloroplast is called the stroma. Within the stroma are stacks of thylakoids, the sub-organelles, which are the site of photosynthesis. The thylakoids are arranged in stacks called grana. A thylakoid has a flattened disk shape. Inside it is an empty area called the thylakoid space or lumen. Photosynthesis takes place on the thylakoid membrane. 4|Page 1.1.3.7 Cell Wall The cell wall gives a definite shape and provides proection to the protoplasm. It is non-living in nature and and is permeable. Adjacent cells in the plant body remain inter connectd by plasmaodesmata. The cell wall of plants is made of fibrils of cellulose embedded in a matrix of several other kinds of polymers such as pectin and lignin which provide mechanical strength to the cell. Cell wall consists of 3 parts: i) Middle lamella-made of pectic acid in the form of Ca and Mg salts. ii) Primary wall-made of cellulose iii) Secondary wall-pronounced in dead cells, viz tracheids and sclerenchyma. 1.1.3.8 Other organelles i.Ribosome: Ribosome is a small organelle composed of RNA-rich cytoplasmic granules that are sites of protein synthesis. Each ribosome has a large and a small subunit. Each subunit contains about 65% RNA and 35% protein. ii.Lysosome: Lysosome is round organelles surrounded by a membrane and containing digestive enzymes. Lysosomes function in the digestion of materials brought into cell by phagocytosis or pinocytosis. They also serve to digest cell components after cell death. Golgi iii.The Golgi apparatus: The Golgi apparatus consists of Body flattened, single membrane vesicles. Some become vacuoles in which secretary products are concentrated. The primary function of the Golgi apparatus is to process and package macromolecules, such as proteins and lipids, after their synthesis and before they make their way to their destination. It is important in the processing of proteins for secretion. It has a system of outer flattened cisternae which appear as roughly parallel membranes enclosing a space 60- 90Ǻ with a distance of about 200Ǻ between them. iv. Vacuole: A vacuole is a membrane bound organelle which is present in all plant and fungal cells and some animal and bacterial cells. From the outside, the vacuole is surrounded by tonoplast.Vacuoles are essentially enclosed compartments which are filled with water containing inorganic and organic molecules including enzymes in solution, though in certain cases they may contain solids which have been engulfed. Vacuoles are formed by the fusion of multiple membrane vesicles and are effectively just larger forms of these. The functions of the vacuole include: isolation of materials that might be harmful or a threat to the cell, containing waste products, exporting unwanted substances from the cell, maintaining internal hydrostatic pressure or Turgot within the cell and maintaining internal pH. Unit 2: Major molecules Chapter 1: Major molecules in foods and their important functions 2.1.1 Major molecules in food The four major classes of molecules in food are carbohydrates, protein, fat and nucleic acids. They are made up of relatively small micromolecules called monomers that are linked together to create large macromolecules, which are known as polymers. When monomers are linked together to synthesize a biological polymer, they undergo a process called synthesis. 2.1.2 Functions of macromolecules 1. To provide materials for growth and repair of tissues in order to provide and maintain the basic structure of our bodies. 2. To supply the body with energy required to perform external and internal activities 5|Page  3. To provide materials which regulate the functions of the body. 4. To store and decode genetic information 5. To perform structural and catalytic roles in cells 2.1.3 Nature of Major molecules The four main classes of molecules present in food are carbohydrates, lipids, protein, and nucleic acids. These molecules are called macromolecules or polymers. They are made up of small micromolecules, called monomers that are linked to form these polymers. Monomers are linked together to synthesize a biological polymer by enzyme catalysed reactions. 2.1.3.1 Carbohydrates Carbohydrates are the most widely distributed and abundant organic compound on earth. They play an important role in the metabolism of animals and plant. Carbohydrate biosynthesis in plants starting from carbon dioxide and water with the help of light energy (Photosynthesis) is the basis for the existence of all other organisms which depend on the intake of organic substances with food. Functions: They are the major source of biological energy through their oxidation in the tissues.They also furnish organic precursors for the biosynthesis of many cell components. Sources: Carbohydrate rich foods are abundant and cheap compared with fats and protein. Food carbohydrates are starches and sugars found in cereal grains, tubers, vegetables, legumes, fruits, milk and milk products. Fructose is present in honey. Fiber, consisting largely of cellulose and other non digestible cell-wall polymers of plant origin plays no metabolic role but helps to maintain proper motility in the intestinal tract. 2.1.3.2 Lipids Lipids refer to substances such as fats and oils and fat like substances present in food. They are another group of major molecules found in all cells. Lipids exhibit greater structural variety than the other classes of biological molecules. Functions: In the form of lipid bilayers they are essential components of biological membranes. Lipids containing hydrocarbon chains serve as energy stores. Many intra-and intercellular signaling events involve lipid molecules. In addition to triglycerides, cholesterol and phospholipids provide structure to the body cells. Sources: Important animal sources of fat are meat, milk, eggs and fish and important plant sources are all oils and vanaspathy. Fats from animal sources are relatively rich in saturated fatty acids but contain a low content of polyunsaturated acids. Plant fats and fish are rich in polyunsaturated fatty acids. Cholesterol is present in significant amounts in animal products, such as egg yolk, butterfat, and meat. It is absent in plant foods. 2.1.3.3 Proteins Proteins are the most abundant substances in all cells forming about 50% of a cell's over all mass. A protein is an unbranched polymer of amino acids. Functions: Proteins supply the required amino acids as building blocks for protein biosynthesis. Amino acids are precursors of other nitrogen containing substances such as enzymes, hormones, porphyrins, and many other biomolecules. Oxidation of the carbon skeletons of amino acids also furnishes significant fraction of the total daily energy requirement. They also contribute to the flavour of food and are precursors of aroma compounds and colours formed during thermal or enzymatic reactions in production, processing and storage of food. Sources: Meat, fish, milk, egg, pulses, beans and peas are important sources of protein. 2.1.3.4 Nucleic acids Nucleic acids are polymers composed of nucleotide monomers. The Nucleotides are made up of a five-carbon sugar, a heterocyclic 6|Page nitrogenous base and phosphate group(s). They are joined to one another by covalent bonds between the phosphate of one and the sugar of another. These linkages are called phosphodiester linkages. There are two types of nucleic acids found within cells: deoxyribonucleic acid (DNA) and ribonucleic acid(RNA). Nearly all the DNA is found within the cell nucleus. It is repository of hereditary character. RNA occurs in all parts of a cell. It is essentially required for protein biosynthesis. Function - The primary function of DNA is the storage and transfer of genetic information. This information is used (indirectly) to control many functions of a living cell. In addition, DNA is passed from existing cells to new cells during cell division. RNA functions primarily in the synthesis of proteins, the molecules that carry out essential cellular functions. Unit 3: Carbohydrates Chapter 1: Classification and functions of Carbohydrates 3.1.1 Carbohydrates Carbohydrates are the most abundant class of macromolecule on earth. They constitute about 75% by mass of dry plant material. The plants produced carbohydrate via photosynthesis. In this process, glucose is synthesized from carbon dioxide and water by photosynthesis and stored as starch or converted to cellulose in the plant framework. Animals can synthesize some carbohydrate from fat and protein, but the bulk of animal carbohydrate is derived ultimately from plants. They are organic compounds that contain carbon, hydrogen, and oxygen. Generally the hydrogen and oxygen in carbohydrates are present in the ratio of two hydrogen to one oxygen (2:1) as in water H2O hence the term carbohydrates (carbon hydrate) were derived. But all compounds grouped under this heading have a similar structure. The simple sugar glucose contains 6 carbon atoms, 12 hydrogen atoms, and 6 oxygen atoms giving the molecular formula C6H12O6. Carbohydrates are derivatives of polyhydoxy aldehydes or ketone. Functions: Carbohydrate oxidation provides energy. They are stored as glycogen in animal tissues and serve as short-term energy reserve. They supply carbon atoms for the synthesis of other biochemical compounds- proteins, lipids and nucleic acids. Carbohydrate form part of the structural component of DNA and RNA molecule. 3.1.2 Classification The carbohydrates are often referred as saccharides named after the Greek word ‘Sakcharon’, meaning sugar. They are classified as follows. 1) Monosaccharide 2) Disaccharide 3) Oligosaccharide 4) Polysaccharide 3.1.2.1 Monosaccharide Type of Source Functions pentose D-Ribose Nucleic acids Structural elements in nucleic acids and coenzymes, e.g. ATP, NAD, NADP, flavoprotein. Ribose- phosphate is an intermediate in pentose phosphate pathway. D-Ribulose Formed in metabolism Ribulose phosphate is an intermediate in pentose phosphate path way. D-Arabinose Gum arabic, plum and cherry Constituent of glycoproteins gums. D-Xylose Wood gums, proteoglycans, Constituent of glycoproteins glycosaminoglycans 7|Page  Type of Hexose Sourse Functions D-Glucose Fruit juices. Hydrolysis of The free sugar of the body. The sugar starch, cane sugar maltose, carried by the blood, and the principal one and lactose. used by the tissues. D-Fructose Fruit juices, It can be changed to glucose in the liver and thus Honey, Hydrolysis of cane used in the body. sugar. D-Galactose Hydrolysis of lactose. It can be changed to glucose in the liver and metabolized. It is synthesized in the mammary gland to make the lactose of milk. It is a constituent of glycolipid and glycoprotein. D-Mannose Hydrolysis of plant It is a constituent of many glycoproteins. mannan(plant polysaccharides) and gums. A monosaccharide is a carbohydate that contains a single polyhydroxy aldehyde or polyhydroxy ketone unit. Monosachharides can not be hydrolyzed to simpler carbohydrates. They may be subdivided into trioses, tetroses, pentoses, hexoses or heptoses depending upon the number of carbon atoms they posses. Pentoses and hexoses are important simple sugars present in food. 3.1.2.2 Disaccharide A disaccharide is a carbohydrate that contains two molecules of monosaccharide units covalently bonded to each other. Examples: Maltose, lactose and sucrose. On hydrolysis, maltose yields two molecules of glucose, lactose yields one molecule of galactose and one molecule of glucose, and sucrose yields one molecule of glucose and one molecule of fructose. Maltose is formed when starchy material is hydrolysed. Sucrose is present in fruit juices, sugarcane and sugar beet. Lactose is the principal sugar present in milk. 3.1.1.3 Oligosaccharide An oligosaccharide is a carbohydrate that contains two to ten monosaccharide units covalently bonded to each other. Raffinose and stachyose are two important oligosaccharides present in dried beans, peas and lentils, which yield three and four monosaccharide units respectively on hydrolysis. 3.1.1.4 Polysaccharides A polysaccharide is a polymeric carbohydrate chain that contains more than ten molecules of monosaccharide units covalently bonded to each other. E.g.Gycogen present in liver and muscle cells of animals, Starches, cellulose and dextrins in plant tissues. These are sometimes designated as hexosans or pentosans, depending upon the identity of the monosaccharides they yield on hydrolysis. These may be classified as homopolysaccharides and heteropolysaccharides depending on the presence of either the same monosaccharids or more than one monosaccharide. E.g. Homopolysaccharide- Starch, Glycogen, cellulose, Dextrin Heteropolysaccharide- Mucopolysaccharide 8|Page 3.2.1 Structure of Carbohydrates The name carbohydrate owes its origin to the fact that most substances of this class have empirical formulas suggesting they are carbon ”hydrates” in which the ratio of carbon to hydrogen to oxygen atoms is 1:2:1. For example, the empirical formula is of D-glucose is C6H12O6. This also can be written as (CH2O)6 or C6(H2O)6. Many carbohydrates conform to the empirical formula (CH2O)n while others do not show this ratio. 3.2.1.1 Structure of Monosaccharides Monosaccharides are polyhydric alcohols containing aldehyde or keto groups. They may be classified into aldoses or ketoses depending upon whether the aldehyde or ketone groups are present. Depending upon the number of carbon atoms they divided into trioses, tetroses, pentoses, hexoses or heptoses. 3.2.1.1.1 Stereochemistry Stereochemistry is the study of the arrangement of atoms in three- dimensional space. Stereoisomer is compounds in which the atoms are linked in the same order but differ in their spatial arrangement. Many carbohydrates exhibit stereoisomerism as they contain the same number of atoms and the same kinds of group but have different chemical and biological properties. This is due to the presence of asymmetric carbon atom in the structure. A carbon atom attached to four different atoms or groups is called an asymmetric carbon atom. When there are many asymmetric carbon atom in a chain molecule the number of stereoisomer possible is equal to 2n, where n is the number of asymmetric carbon atoms. The simple sugars corresponding to the formula C6H12O6 have 4 asymmetric carbon atoms and hence have 24 = 16 stereoisomer each have identical functional groups but with different spatial configurations. This is due to the presence of asymmetric carbon atom in the structure. Glucose has 4 asymmetric carbon atoms (2, 3, 4 & 5). The structure of glucose can be represented in three ways (1) the straight - chain structural formula (aldohexose, as simple ring or a chair form. 3.2.1.1.2 Isomerism Monosaccharide exhibits various forms of isomerism due to the presence of asymmetric carbon atom. There are six types of isomerism found with monosaccharides. 1. Aldose-ketose isomerism 4. Pyranose and furanose isomerism 2. D&L isomerism 5. Alpha and Beta isomerism 3. Optical isomerism 6. Epimerism 3.2.1.1.2.1 Aldose -Ketose Isomerism Monosaccharides exhibit aldose-ketose isomerism. Sugar with a keto groups is called a ketose and that with an aldehyde group is called an aldose. Glucose is an aldose since there is a potential aldehyde group in position 1 of glucose. Fructose is a ketose since it has a potential keto group in position 2. Fructose has the same molecular formula as glucose but differs in its structural for 3.2.1.1.2.2 D&L Isomerism The designation of a sugar isomer as the D form or of its mirror image as the L form is determined by its spatial relationship to the parent compound of the carbohydrate family, the three carbon sugar glyceraldehydes. 9|Page  The orientation of the -H and -OH groups around the carbon atom adjacent to the terminal primary alcohol carbon determines whether the sugar belongs to the D or L series. When the -OH group on this carbon is on the right, the sugar is a member of the D series; when it is on the left, it is a member of the L series. Most of the monosaccharide occurring in nature is of the D configuration. 3.2.1.1.2.3 Optical Isomerism One of the physical properties of molecules that have asymmetric carbon atoms is that the molecules will rotate the direction in which the light is vibrating as it passes through them. The presence of asymmetric carbon atoms confers optical activity on the compound and exhibit optical isomerism. 'When a beam of plane -polarized light is passed through a solution of an optical isomer, it will be rotated either to the right-or left in accordance to the type of compound and it is called optical activity' A compound which causes rotation of plane polarized light to the right is said to be dextrorotatory (+) isomer, and 'd' is used to designate the fact. Rotation of the plane polarised light to the left is called levorotatory isomer and designated by a (-) sign and 'l' is used to designate to the fact. The naturally occurring form of fructose is the D (-) isomer. When equal amounts of D and L isomers are present, the resulting mixture has no optical activity, since the activities of each isomer cancel one another. Such a mixture is said to be a racemic or DL mixture. Two important monosaccharides whose isoforms are biologically active –L fucose and L iduronic acid. Stereoisomerism and optical activity are independent properties. Thus D (+), D (-), L (-), or L (+), indicate structural relationship to D or L glyceraldehyde and the optical activity exhibited.. Synthetically produced compounds are racemic.The optical rotation of glucose in solution is dextorotation hence, the alternative name of dextrose. 3.2.1.1.2.4 Pyranose and Furanose Ring Structures Alcohols react with the carbonyl groups of aldehydes and ketones to form hemiacetals and hemiketals respectively. The hydroxyl and either the aldehyde or the ketone groups of monosaccharides can likewise react intramolecularly to form cyclic hemiacetals and hemiketals. The configurations of the substitutes of each carbon atom in these sugar rings are conveniently represented by their Haworth's Projection, in which the heavier ring bond project in front of the plane of the paper and the lighter ring bonds project behind it. A sugar with a six-member ring is known as a pyranose in analogy with pyran, the simplest compound containing such a ring. Similarly, sugars with five- member rings are furanoses in analogy with furan. Glucose and fructose form pyranose and furanose ring structures. Other pentoses and ketoses may also show ring structures. Ring structures of sugars presented are the most stable structures present in natural compounds. 10 | P a g e 3.2.1.1.2.5 Alpha and Beta Anomers When a monosaccharide cyclizes, the carbonyl carbon, called the anomeric carbon, becomes a chiral center with two possible configurations. The pair of stereoisomers that differ in configuration at the anomeric carbon are called anomers. In the α anomer, the -OH substitutent of the anomeric carbon is on the opposite side of the sugar ring from the - CH2OH group at the chiral center that designates the D or L configuration(C5 in hexoses). The other form is known as the ß anomer. The two anomers of D-glucose have slightly different physical and chemical properties, including different optical rotations. The anomers freely interconvert in aquous solution, so at equilibrium, D- glucose is a mixture of the ß anomer (63.6%) and the α anomer (36.4%). The linear form is normally present in only minute amounts. 3.2.1.1.2.6 Epimerism Epimers are diastereoisomers whose molecules differ only in the configuration at one chiral center. Isomers differing as a result of variations in configuration of the -OH and -H on carbon atoms 2, 3 and 4 glucose are known as epimers of glucose. The most important epimers of glucose are mannose and galactose, formed by epimerization at carbons 2 and 4, respectively. 3.2.2 Monosaccharide derivatives Monosaccharide units in which an OH group is replaced by other groups are called as monosaccharide derivatives. 1. Glycoside 2. Amino sugar 3. Deoxy sugars 3.2.2.1 Glycosides Glycosides are compounds formed by the condensation reaction between the hydroxyl group of a sugar and the hydroxyl group of a second compound (aglycon) which may or may not be another sugar. A glycoside produced from glucose is called a glucoside, that from galactose is called a galactoside, and so on. Glycosides like the hemiacetals from which they are formed, can exist in both α and ß forms.Glycosides are named by listing the alkyl or aryl group attached to the oxygen, followed by the name of monosaccharide involved, with the suffix-ide appended to it. 11 | P a g e 3.2.2.2 Amino sugars (Hexosamines) An amino sugar is formed if one of the hydroxyl groups of a monosaccharide is replaced with an amino group. In naturally occurring amino sugars, of which there are three common ones, the amino group replaces the carbon 2 hydroxyl group. The three common natural amino sugars are D-Glucosamine, D- Galactosamine and D-Mannosamine. Amino sugars and their N-acetyl derivatives are important building blocks of polysaccharides found in cartilage and exoskeleton of crustaceans and insects. Glucosamine is a constituent of hyaluronic acid. Chitin is a polymer of N acetyl Glucosamine. Galactosmine is a constituent of glycoprotein and Mannosamine is part of mucoprotein. 3.2.2.3 Deoxy sugars Monosaccharide units in which an OH group is replaced by H are known as deoxy sugars. The biologically most important of these is β-D-2- deoxyribose, the sugar component of DNA’s sugar-phosphate backbone. 3.2.2.4 Oxidation products When aldosed are oxidised under proper conditions with different types of oxidising products, three types of acids are produced. 1. Aldonic acid 2. Uronic acid 3. Saccharic acid 1. Aldonic acid: Oxidation of an aldose with bormine water at neutral pH converts the aldehyde group to a carboxyl group. D Glucose → Gluconic acid 2. Uronic acid: When aldoses are oxidised with H2O2 uronic acids are formed. Uronic acids are constituents of pectic polysaccharides. 3. Saccharic acid or Aldaric acid: When aldoses are oxidised with nitric acid saccharic acids are formed. Glucose forms glucosaccharic acid and galactose produces mucic acid. 12 | P a g e 3.2.3 Properties of Monosaccharides Monosaccharides exhibit different properties due to the presence of aldehyde or ketone groups. Some of these reactions are important for analytical purpose. 3.2.3.1 Reducing action of sugars in alkaline solution All the sugars that contain the free sugar group undergo enolization and various other changes when placed in alkaline solution. The enediol forms of the sugars are highly reactive and are easily oxidized by oxygen and other oxidizing agents. These sugars in alkaline solution are very powerful reducing agents and the sugars are oxidized to complex mixtures of acids. This reducing action of sugars in alkaline solution is utilized for both the qualitative and quantitative estimation of sugars. When a reducing sugar is heated with the alkaline copper reagents, the following reaction occurs: Sugar + alkali → Dienol [Copper complex of tartarate or citrate (Cu ++)] Cu2+ + -OH- + mixture of sugar acids → Cu(OH)2 → Cu2O (red) The Cu2+ ions take electrons from the enediols and oxidize them to sugar acids and are in turn reduced to cuprous ions, Cu+. The cuprous ions combine with hydroxyl ions to the yellow cuprous hydroxide, which upon heating is converted to red cuprous oxide, Cu2O. The appearance of a yellow-to-red precipitate indicates reduction, and the quantity of sugar present can be roughly estimated from the amount of colour. In quantitative determination, the amount of copper reduced is obtained by iodometric titration or colorimetric methods by treatment with reagents, and the amount of sugar is calculated. 3.2.3.2 Action of acids on carbohydrates Polysaccharides in general are hydrolyzed into their constituent monosaccharide by boiling with dilute (0.5- 1.0N) mineral acids, such as hydrochloric or sulfuric. The monosaccharide when treated with strong acids, to furfural and hydroxy methyl furfural. The furfural and hydroxyl methyl furfural content may be quantitatively determined by use of its color reaction with organic compounds. Anthrone is one such compound used for the estimation of sugars which forms a green coloured compound which is measured colorimetrically or spectrometrically. 3.2.3.3 Mutarotation The optical rotation of a freshly prepared solution of glucose gradually decreases and finally becomes constant. This change in the rotation of sugar solutions upon standing is called “mutarotation”. It is a general property of reducing sugars. When D-glucose is crystallized from water or dilute alcohol at room temperature, a form 13 | P a g e separates having an initial specific optical rotation of +112o which changes to +52.5o. If, it is crystallized from water at temperatures above 98oC, a different form of glucose, having an initial specific rotation of +19o which changes to +52.5o, is obtained. The first of these isomers of glucose was called “α-D-glucose” and the second “β-D-glucose”. Glucose exists in different isomeric forms in solution which change into the same equilibrium mixture regardless of which form is dissolved. Chapter 3: Structure and function of Polysaccharides 3.3.1 Structure of Disaccharides The disaccharides are sugars composed of two monosaccharide residues united by a glycosidic linkage. Their chemical name reflects their component monosaccharide. The important disaccharides of food are maltose, sucrose, and lactose. a. Structure of Sucrose - Sucrose, common table sugar, is the most abundant of all disaccharides and occurs throughout the plant kingdom. It is made up of one glucose unit and a fructose unit linked by α 1-2 β glycosidic bond between the aldehyde or keto groups. Hence it is a nonreducing sugar. Sucrose occurs in the juices of sugar beets, sugar cane, sorghum, sugar maple and juices of pineapple and other fruits. Ripe fruits are rich in sucrose. It is the most abundantly distributed sugar. Invert sugar - Hydrolysis of sucrose yields a crude mixture called “invert sugar” because the optical activity of dextro rotatory sucrose (+66.5o) changes (inverts )to -19.5o due to the production of the strongly levorotatory fructose. Because of the inversion of the sign of rotation in the reaction, the process is referred to as “inversion”, and the mixture of glucose and fructose obtained is called “invert sugar”. Honey contains a large proportion of invert sugar. b. Structure of Maltose - Maltose is composed of two glucose units linked by α 1-4 glycosidic bond bond. It is formed when starch is hydrolysed by the action of salivary amylase (ptyalin) and pancreatic amylase during the processes of digestion. Maltose is also formed as an intermediate product in the acid hydrolysis of starch and consequently is an important constituent of corn syrups, which are prepared by partial hydrolysis of starch with dilute acid. These syrups are complex mixtures of dextrins, maltose, and glucose. Commercial malt sugar is a mixture of maltose and dextrins formed from starch by hydrolysis with amylase. c. Structure of Lactose - Lactose is the milk sugar and it is made up of galactose and glucose linked by ß (1-4)glycosidic bond. Lactose is formed by 14 | P a g e the mammary glands and occurs to the extent of about 5 per cent in milk. It has a free aldehyde group and hence shows mutarotation and the final specific rotation is +55.2º d.Trehalose: It is anonreducing disaccharide, occurs as a major constituent of the ciculating fluids (hemolymph) of insects and serve as energy storage compound. It is made of two glucose molecules in which the anomeric carbon of these units is linked by glycosidic bond. 3.3.2 Structure of Oligosaccharide Raffinose, also called melitose, is a trisaccharide that is widely found in legumes and cruciferous vegetables, including beans, peas, cabbage, brussels,sprouts, and broccoli. It consists of galactose connected to sucrose via a α 1→6 glycosidic linkage. Humans cannot digest polysaccharides with this linkage and the trisaccharides are fermented in the large intestine by gas-producing bacteria. Stachyose is a tetrasaccharide containing glucose, fructose and two galactose units. It occurs in legumes and dry beans and peas. 3.3.3 Structure of polysaccharides Polysaccharides consist of long chains having hundred or thousands of monosaccharides units bonded to each other by glycosidic linkages. Polysaccharides are often also called glycans. They are classified as homopolysaccharides or heteropolysaccharides if they consist of one or more types of monosaccharide. Some polysaccharides such as cellulose linear chain whereas others, such as glycogen, have branched chains. 3.3.3.1 Starch Starch is a polymer of glucose. It yields only glucose on hydrolysis and hence is a homopolysaccharide and is called a glucosan or glucan. The two chief constituents of starch are amylose (15-20%), which has a non branching helical structure, with glucose units linked by α(1-4)-glucosidic bond in the chains and amylopectin (80-85%), which consists of branched chains composed of 24-30 glucose residues united by α (1-4) glucosidic linkages in the chains and by α (1-6) glucosidic linkages at the branch points. Amylose structure given below 15 | P a g e Structure of Amylopectin Starch is the most important food source of carbohydrate and is found in cereals, potatoes, legumes, and other vegetables. Rich sources of starch are grains such as wheat, rice, corn, oats, millets and barley, legumes such as peas, beans and lentils and tubers such as potatoes, yam and cassava. 3.3.3.2 Glycogen Glycogen is a homopolysaccharide containing only glucose units. Glycogen is the storage polysaccharides of the animals. It is often called animal starch. It is a highly branched structure similar to amylopectin and has chains of 12-14 α-D-glucopyranose residues (in α[1-4]- glucosidic linkage) with branching by means of α(1-6 ) - glucosidic bonds. Skeletal muscle and the liver are the two important sites of glycogen storage. 3.3.3.3 Dextrins Dextrins are substances formed in the course of the hydrolytic break down of starch. Limit dextrins are the first formed products as hydrolysis reaches certain degree of branching. They form sticky solutions is water and are used as adhesive. 16 | P a g e 3.3.3.4 Cellulose Cellulose is the structural component of plant cell walls, it the most abundant naturally occuring polysachharide. It is insoluble and consists of D-glucopyranose units linked by ß(1-4) glycosidic bonds to form long, straight chains strengthened by cross-linked hydrogen bonds. Cellulose cannot be digested by many mammals, including humans, because of the absence of an enzyme that attacks the β linkages. But, it is an important source of “bulk” in the diet. In the stomach of ruminants and other herbivores, there are microorganisms that can attack the β linkage, making cellulose available as a major calorigenic source. 3.3.3.5 Chitin Chitin is a polysachharide that is similar to cellulose in both function and structure. Structurally, chitin consists of N - acetyl - D- glucosamine units joined by ß(1-4)- glycosidic bond. Its function is to give rigidity to the exoskeletons of crabs, lobsters, shrimp, insects , and other arthropods. It also occurs in the cell walls of fungi. 3.3.3.6 Pectins Pectin occurs widespread in nature. Pectin is combined with cellulose in the cell walls. The combined, insoluble pectin is referred to as proptopectin. It may be released by mild hydrolysis or other means and converted into soluble pectin. When soluble pectin is boiled with dilute acid, it is slowly hydrolyzed to pectic acid and methyl alcohol. Purified pectin accordingly is the methyl ester (many methyl groups per molecule) of pectic acid. Pectic acid is a chain of at least 200 of (1-4) linked β-D galactopyranosyl uronic acid units. It is found especially in the pulp of citrus fruits, apples, beets and carrots Uses:Pectin is used commercially in the preparation of jellies, jams, and marmalades. Its thickening properties also make it useful in the confectionery, pharmaceutical, and textile industries. Pectin also has several health benefits in humans.Included among these are its ability to reduce low density lipoprotein (LDL) levels, thereby lowering cholesterol levels, and its ability to slow the passage of food through the intestine, relieving diarrhea. Pectin can also activate cell death pathways in cancer cells, indicating that pectin may play an important role in preventing certain types of cancer. 3.3.4 Seaweed polysaccharides Significant amounts of seaweed polysaccharides are used in food, pharmaceuticals and other products for human consumption. Commercially important are, 1. Agar 2. Agarose 3. Carageenan 4. Alginic acid 17 | P a g e 3.3.4.1 Agar Agar is a polymer made up of subunits of the sugar galactose.The word agar comes from the Malay word agar-agar (meaning jelly). It is also known as kanten or agal- agal (Ceylon agar). Structure: It is an unbranched polysaccharide obtained from the cell walls of some species of red algae or seaweed. Agar polysaccharides serve as the primary structural support for the algae's cell walls. Uses: Gracilaria, Gelidium, Pterocladia and other red algae are used in the manufacture of the all- important agar. Agar can be used as a vegetarian gelatin substitute, a thickener for soups, in jellies, ice cream and desserts, as a clarifying agent in brewing, a laxative and for paper sizing fabrics. It is used widely as a growth medium for microorganisms and for microbiological and biotechnological applications. 3.3.4.2 Agarose Agar is a heterogeneous mixture of two classes of polysaccharide: agaropectin and agarose. Although both polysaccharide classes share the same galactose-based backbone, agaropectin is heavily modified with acidic side-groups, such as sulfate and pyruvate. The neutral charge and lower degree of chemical complexity of agarose make it less likely to interact with biomolecules, such as proteins. Uses: Gels made from purified agarose have a relatively large pore size, making them useful for size- separation of large molecules, such as proteins or protein complexes >200 kilo Daltons, or DNA fragments >100 base pairs. Agarose can be used for electrophorotic separation in agarose gelelectrophoresis or for column-based gel filtration. 3.3.4.3 Carrageenan Carrageenans or carrageenins are a family of linear sulphated polysaccharides extracted from red seaweeds. The name is derived from a type of seaweed that is abundant along the Irish coastline near the village of Carragheen. Carrageenan consists of alternating 3-linked-β-D-galactopyranose and 4-linked-α-D-galactopyranose units. Carrageenan is a collective term for polysaccharides prepared by alkaline extraction (and modification) from red seaweed (Rhodophycae), mostly of genus Chondrus, Eucheuma, Gigartina and Iridaea. Different seaweeds produce different carrageenans. Uses: Carrageenans are used mainly for thickening, suspending and gelling. κ- and ι-carrageenans form thermoreversible gels on cooling in the presence of appropriate counterions. κ-Carrageenan stabilizes milk -casein products due to its charge interaction with the casein micelles (~200 nm diameter); their incorporation into the network preventing whey separation. Such complexes are soluble when both have same charge and are held together by counterions or oppositely charged patches. Carrageenan is also used as a binder in cooked meats, to firm sausages and as a thickener in toothpaste and puddings. 3.3.4.4 Alginic Acids Alginic acid is a linear copolymer with homopolymeric blocks of (1-4)- linked ß-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues, respectively, covalently linked together in different sequences or blocks. It is (algine, alginate) is a viscous gum that is abundant in the cell walls of brown algae. Brown Seaweeds are used for extraction of alginic acid. Commercial varieties of alginate are extracted from brown seaweed, including the giant kelp Macrocystis pyrifera, Ascophyllum nodosum and various types of Laminaria, Sargassum sp, Turbinaria ornata, Padina sp etc. Uses: Alginic acid is used in the food industry, for thickening soups and drinks, ice cream, tinned meats and jellies. Alginate absorbs water quickly, which makes it useful as an additive in dehydrated products such as slimming aids, and in the manufacture of paper and textiles is used commercially for toothpastes, soaps, fabric printing, and a host of other applications. It forms a stable viscous gel in water, and its primary function in the above applications is as a binder, stabilizer, emulsifier, or moulding agent. It is also used for waterproofing and fireproofing fabrics, as a gelling agent, and cosmetics, and as a detoxifier 18 | P a g e that can absorb poisonous metals from the blood. Alginate is also produced by certain bacteria, notably Azotobacter species. Chapter 4:Intermediary Metabolism and carbohydrate digestion 3.1.14.Intermediary metabolism The intermediate steps within the cells in which the nutrient molecules or foodstuffs are metabolized and converted into cellular components catalyzed by enzymes is called Intermediary metabolism. The reaction pathways that comprise metabolism are divided into two categories. i) Catabolism: Pathways involved in degradation such as oxidative processes that release free energy in the form of high energy phosphates (ATP) or reducing equivalents E.g Respiratory chain and oxidative phosphorylation. In catabolic reactions, complex substances are broken down to simpler compounds with a concomitant release of free energy, the released free NADPH. The major nutrients such as carbohydrates, lipids and proteins are converted to common intermediate and further metabolized in a central oxidative pathway. ii) Anabolism: Pathways involved in biosynthesis e. g. Biosynthesis of proteins, lipids etc. Simple organic molecules such as pyruvic acid, acetyl unit or intermediate compounds of citric acid cycle serve as starting molecules for varied biosynthetic products. The energy rich molecules such as ATP or NADPH derived from catabolic reactions are utilized in the biosynthetic reactions. 3.4.1.5.Basic metabolic pathways The basic metabolic pathways that process the major products of digestion: The nature of the diet sets the basic pattern of metabolism in the tissues. Mammals such as humans need to process the absorbed products of digestion of dietary carbohydrate, lipid, and protein. These are mainly glucose, fatty acids and glycerol, and amino acids, respectively. In ruminants (and to a lesser extent other herbivores), cellulose in the diet is digested by symbiotic microorganisms to lower fatty acids (acetic, propionic, butyric), and tissue metabolism in these animal is adapted to utilize lower fatty acids as major substrates. All these products of digestion are processed by their respective metabolic pathways to a common product, acetyl- CoA, which is then completely oxidized by the citric acid cycle (Fig 4.1). 3.4.1.3.Carbohydrate metabolism 1. Carbohydrate metabolism - It is centered on the provision and fate of glucose. Glucose is metabolized to pyruvate and lactate in all mammalian cells by the pathway of glycolysis. Glucose is a unique substrate because glycolysis can outline of the pathways for the catabolism of dietary carbohydrate, protein, and fat. All the pathways lead to the production of acetyl-CoA, which is oxidized in the citric acid cycle, ultimately yielding ATP in the process of oxidative phosphorylation. Occur in the absence of oxygen (anaerobic)are able to 19 | P a g e metabolize pyruvate to acetyl-CoA, which can enter the citric acid cycle for complete oxidation to CO2 and H2O, with liberation of much free energy as ATP in the process of oxidative phosphorylation. Glucose is a major fuel of many tissues. But it (and some of its metabolites) also takes part in other processes, e.g. (1) Glycogenesis- the conversion to its storage polymer, glycogen, particularly in skeletal muscle and liver. (2) the pentose phosphate pathway, which arises from intermediates of glycolysis. It is a source of reducing equivalents (2H) for biosynthesis e.g., of fatty acids-and it is also the source of ribose, which is important for nucleotide and nucleic acid formation. (3) Triose phosphate gives rise to the glycerol moiety of acylglycerols (fat). (4) Pyruvate and intermediates of the citric acid cycle provide the carbon skeletons for the synthesis of amino acids, and acetyl-CoA is the building block for long-chain fatty acids and cholesterol, the precursor of all steroids synthesized in the body. (5)Gluconeogenesis is the process that produces glucose from non-carbohydrate precursors, e.g. lactate, amino acids, and glycerol. 3.4.1.4.Lipid metabolism Lipid metabolism is concerned mainly with fatty acids and cholesterol.The source of long-chain fatty acids is either dietary lipid or de novo synthesis from acetyl-CoA derived from carbohydrate. In the tissues, fatty acids may be oxidized to acetyl-CoA (β-oxidation) or esterified to acylglycerols, where, as triacylglycerol (fat) they constitute the body’s main caloric reserve. Acetyl-CoA fromed by β-oxidation has several important fate 1) In the case of acetyl-CoA derived from carbohydrateit is oxidised completely to CO2 + H2O via the citric acid cycle. Fatty acids yield considerable energy both in β-oxidation and in the citric acid cycle and are 20 | P a g e therefore very effective tissue fuels. (2) It is a source of the carbon atoms in cholesterol and other steroids. (3) In the liver, it forms ketone bodies, alternative water-soluble tissue fuels that become important sources of energy under certain conditions (eg, starvation) 3.4.1.5.Amino acid metabolism Much of amino acid metabolism involves transamination.The amino acids are necessary for protein synthesis. Some must be supplied specifically in the diet (the essential amino acids), since the tissues are unable to synthesize them. The remainder, or nonessential amino acids, are also supplied in the diet, but they also can be formed form intermediates by transamination using the amino nitrogen from other surplus amino acids. After deamination, excess amino nitrogen is removed as urea, and the carbon skeletons that remain after transamination (1)are oxidized to CO2 via the citric acid cycle, (2) form glucose (gluconeogenesis), or (3) form ketone bodies. In addition to their requirement for protein synthesis, the amino acids are also the precursors of many other important compounds, eg, pruines, pyrimidines, and hormones such as epinephrine and thyroxine. 21 | P a g e 3.4.2. Digestion, Absorption and Metabolism of Carbohydrates The most abundant carbohydrates ingested by human beings are the polysaccharides, starch and cellulose, furnished by plant foods and glycogen, provided by foods of animal origin. The energy needed to run the human body is obtained from ingested, digested and absorbed food through a multistep process that involves several different catabolic pathways. The digestion, begins in the mouth, continues in the stomach, and is completed in the small intestine. The end products of digestion -glucose and other monosaccharides from carbohydrates are absorbed into the blood, carried to the liver and transported to the body's cells. 3.4.2.1. Digestion Starch and glycogen are completely hydrolyzed by enzyme action in the gastrointestinal tract to yield free D-glucose. This process begins in the mouth during chewing, through the action of amylase (Ptyaline) secreted by the salivary glands. Salivary amylase hydrolyzes many of the α (1–4) glycosidic linkages of starch and glycogen to yield a mixture of maltose, glucose and oligosaccharides. Ptyaline action continues in the swallowed food bolus until gastric HCl inactivates it near pH 4.0. Starch-------------→ Maltose + Oligosaccharides + monosaccharides (Enzyme -- Amylase from saliva, Pancreas and intestinal juices) The digestion of digestible polysaccharides to yield D-glucose is continued and completed in the small intestine, largely by the action of pancreatic amylase, made by the pancreas and secreted via the pancreatic duct into the upper portion of the small intestine. This segment of the small intestine in which most of its digestive activity occurs, is called the duodenum. Disaccharides are hydrolyzed by enzymes located in the outer border of the epithelial cells lining the small intestine. Sucrose is hydrolyzed to D-glucose and D-fructose by sucrase, also called invertase: lactose is hydrolyzed to D-glucose and D-galactose by lactase or β-galactosidase and maltose is hydrolyzed by maltase yielding two molecules of D-glucose. Maltose → two molecules of glucose (enzyme – maltase) Lactose → glucose and galactose (enzyme – lactase) Sucrose → glucose and fructose (enzyme -- sucrase) 3.4.2.2 Absorption The interior surface of the small intestine is composed of micro villi that dramatically enlarge its absorptive surface, accounting for an extraordinary efficiency in absorbing consumed energy substrates. 98 percent of all digestible carbohydrate is absorbed. The digestion of food polysaccharides, such as starch, sucrose and lactose produces the monosaccharides glucose, fructose, and galactose, which pass into the blood stream. Galactose and fructose are converted to glucose in the liver. The liver stores the glucose as glycogen and releases glucose as and when needed to maintain blood glucose level. 3.4.2.3 Effect of Harmone on Glucose metabolism The metabolism of carbohydrates is regulated by a variety of hormones and other molecules. Some of these have already been mentioned in previous sections. The proper functions of the body are dependent on precise control of the glucose concentration in the blood. The normal fasting level of glucose in the blood is 70-90 mg/100 ml. 22 | P a g e Hyperglycemia - If the concentration of glucose in blood is too high (above 120 mg/100 mL) a condition known as hyperglycemia results. Hyperglycemia may temporarily exist as a result of eating a meal rich in carbohydrates. Hypoglycemia - If the concentration of glucose is too low (below 70 mg/100 ml) a condition of hypoglycemia exists. Hypoglycemia is characterized by general weakness, trembling, drowsiness, headache, profuse perspiration, rapid heart beat, and possible loss of consciousness. Insulin - The major effect of insulin is to promote the transport of sugar across the cell membrane of fat and muscle cells. In addition, insulin promotes anabolic processes such as increasing the rate of synthesis for glycogen ( glycogenesis), fatty acids, and proteins. Insulin inhibits the catabolic processes such as the breakdown of glycogen and fat. A deficiency of insulin (hypoinsulinism) results in a permanent hyperglycemic condition known as diabetes mellitus. If little or no insulin is present, glucose cannot be utilized properly by the cells and accumulates in the blood. Fatty acid metabolism is also upset. Glucagon - Glucagon increases glucose levels in the blood by stimulating the breakdown of glycogen (glycogenolysis) in the liver into glucose which leaves the liver cells and enters the blood stream. The method of hormone stimulation is a complex cascade effect. Einephrine (adrenaline) also works in a similar fashion. 3.5.1 Glycolysis 3. Glycolysis or Embden- Meyerhoff Pathway is the major pathway for the utilization of glucose for the production of energy and is found in the cytosol of all cells. Glycolysis can function under aerobic and anaerobic conditions. Glucose is converted in to two molecules of pyruvate, chemical energy in the form of ATP is produced, and NADH- reduced coenzymes are produced. Glucose + 2ADP + 2Pi + 2NAD+ → 2Pyruvate + 2ATP + 2NADH + 2H+ + 2 H2O This metabolic pathway takes place in almost all cells. All of the enzymes of the glycolysis pathway are found in the extramitochondrial soluble fraction of the cell, the cytosol. They catalyze the reactions under aerobic and anaerobic conditions. The over all reactions of glycolytic reactions are presented in below. Glycolysis Anaerobic condition : Glucose → Pyruvate → Lactic acid Aerobic condition : Pyruvate → CO2 + H2O Glycolysis is a ten step process in which every step is enzyme catalyzed. Details of individual steps with in the glycolytic pathway are now considered. 3.5.1.1 Step1: Phosphorylation of Glucose Glucose enters into the glycolytic pathway by phosphorylation to glucose 6- phosphate, accomplished by the enzyme hexokinase. However, in liver parenchyma cells and in pancreatic islet cells, this function is carried out by glucokinase. ATP is required as phosphate donor, and it reacts as the Mg-ATP complex. Hexokinase : Glucose + ATP →glucose 6- phosphate The terminal high-energy phosphate of ATP is utilized, and ADP is produced. The reaction is accompanied by considerable loss of free energy as heat and therefore, under physiologic conditions, may be regarded as irreversible. 23 | P a g e Hexokinase is inhibited in an allosteric manner by the product, glucose 6- phosphate. The function of glucokinase is to remove glucose from the blood following digestion and absorption. It is specific for glucose. 3.5.1.2 Step 2: Converstion of Glucose 6 Phosphate to Fructose 6-Phosphate Glucose 6 phosphate is converted to fructose 6-phosphate by phosphoglucose isomerase, which involves an aldose-ketose isomerization. Glucose 6 phosphate ↔ fructose 6-phosphate ( Phosphoglucose isomerase) 3.5.1.3 Step 3: Phosphorlyation of Fructose 6-Phosphate to Fructose 1, 6-Bisphosphate This reaction is catalyzed by the enzyme phosphofructokinase to produce fructose 1, 6-bisphosphate from Fructose 6 phosphate. The phosphofructokinase reaction is irreversible under physiologic conditions.PEK is the enzyme for the principal rate-limiting step to glycolysis. Fructose 6 phosphate+ ATP → Fructose 1, 6-bisphosphate+ ADP (Phosphofructokinase) 3.5.1.4 Step 4: Cleavage of Fructose 1,6-Bisphosphate Fructose 1, 6-bisphosphate is split by aldolase (fructose1, 6-bisphosphate aldolase) into two triose phosphates, glyceraldehyde -3-phosphate and dihydroxyacetone phosphate. Fructose 1, 6-bisphosphate ↔ Glyceraldehyde -3-phosphate + Dihydroxyacetone phosphate (Aldolase) 3.5.1.5 Step 5: Inter conversion of triose phosphates Glyceraldehyde-3-phosphate and dihydroxy acetone phosphate are interconverted by the enzyme phosphotriose isomerase. Glyceraldehyde-3-phosphate ↔ Dihydroxy acetone phosphate (Phosphotriose isomerase) At this stage 2 molecules glyceraldehyde 3-phosphate are formed. Dihydroxyacetone phosphate is also formed from glycerol of fat, which is phosphorylated to glycerol 3 phosphate and then to dihydroxy acetone phosphate. 3.5.1.6 Step 6: Oxidation of Glyceraldehyde 3-Phosphate to 1, 3 Bisphosphoglycerate Glyceraldehyde-3-phosphate is converted to 1,3 bisphosphglycerate by glyceraldehyde 3-phosphate dehydrogenase using NAD+ as the coenzyme. Finally, by phosphorolysis, inorganic phosphate (pi) is added, forming1, 3 bisphosphoglycerate, and the free enzyme. Glyceraldehyde-3-phosphate + NAD → 1,3 bisphosphglycerate + NADH+ H+ (Glyceraldehyde 3-. phosphate dehydrogenase) Energy released during the oxidation is conserved by the formation of a high-energy sulfur group that becomes, after phosphorolysis, a high- energy phosphate group in position 1 of 1, 3 bisphosphoglycerate. 3.5.1.7 Step 7: Transfer of phosphate group from 1, 3 bisphosphoglycerate 1, 3-bisphosphoglycerate is oxidized to 3- phosphoglycerate by phosphoglycerate kinase. This high- energy phosphate is captured as ATP in a further reaction with ADP. 1, 3-bisphosphoglycerate+ ADP→ 3- phosphoglycerate+ATP (Phosphoglycerate kinase) 24 | P a g e Since two molecules of triose phosphate are formed per molecule of glucose undergoing glycolysis, two molecules of ATP are generated at this stage per molecule of glucose. 3.5.1.8 Step 8: Conversion of 3-Phosphoglycerate In the next reaction 3-phosphoglycerate is to 2-Phosphoglycerate converted to 2-phosphoglycerate by the enzyme phosphoglycerate mutase. 3-phosphoglycerate ↔ 2-phosphoglycerate (Phosphoglycerate mutase) 3.5.1.9 Step 9: Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate The subsequent step is catalyzed by enolase that promotes the reversible removal of a water molecule from 2-Phosphoglycerate to form phosphoenolpyruvate. 2-Phosphoglycerate ↔ Phosphoenolpyruvate + H2O (Enolase) 3.5.1.10 Step 10: Conversion of Phosphoenol pyruvate to pyruvate The high-energy phosphate of phosphoenol pyruvate is transferred to ADP by the enzyme pyruvate kinase to generate, at this stage, two molecules of ATP per molecule of glucose oxidized and enolpyruvate is formed. Enolpyruvate formed is converted spontaneousny to the keto form pyruvate. This is an irreversible step. Phosphoenol pyruvate + ADP → Enolpyruvate+ ATP (Pyruvate kinase) Enolpyruvate → Pyruvate 3.5.1.11 Energy production a. Under aerobic condition - Under aerobic condition, pyruvate is taken up into mitochondria, and after conversion to acety-CoA is oxidized to CO2 by the citric acid cycle. The reducing equivalents from the NADH+H+ formed in glycolysis are taken up into mitochondria for oxidation. Two triosephosphates are produced from each molecule of hexose metabolized. Dihydroxyacetone phosphate can be isomerized by isomerase to glyceraldehyde 3 phosphate which is then converted to 1, 3 diphosphoglyceric acid by glyceraldehyde 3 phosphate dehydrogenase system requiring NAD+ and inorganic phosphate. From this high-energy compound and from phosphoenol pyruvate another high energy compound derived from it, two ATP molecules can be obtained from ADP present in the cell. Since two molecules of triose phosphates are undergoing the above reaction totally four ATP molcules of produced. The NADH formed by the dehydrogenation of glyceraldehyde 3 phosphate is then rexoidised to NAD+ by O2 via Electron transport chain of mitochondria and produce 3ATP molecules. Since two molecules of glyceraldehyde 3 phosphate are involved in the above reaction six ATP molecules are produced.However, two ATP molecules are used up in the production of glucose-6-phosphate from glucose and fructose-1, 6- Bisphosphate from fructose-6-phosphate. The net production of ATP is thus only two ATP molecules per mole of glucose during anaerobic glycolysis or fermentation. b. Under anaerobic condition - If anaerobic conditions prevail, the reoxidation of NADH by transfer of reducing equivalents through the respiratory chain to oxygen is prevented. Then, pyruvate is reduced by the NADH+H+ to lactate, the reaction being catalyzed by lactate dehydrogenase. The reoxidation of NADH via lactate formation allows glycolysis to proceed in the absence of oxygen by regenerating sufficient NAD+ for another cycle of the reaction catalyzed by glyceraldehyde-3 phosphate dehydrogenase. 25 | P a g e Pyruvate + NADH + H+ ↔ NAD+ + Lactate (Lactate dehydrogenase) During fermentation, pyruvate is reduced by NADH to ethyl alcohol being catalysed by alcohol dehydrogenase. Pyruvate + NADH + H+ ↔ NAD+ + Ethyl alcohol (Alcohol dehydrogenase) The conversion of two triose phosphates to lactic acid (or ethanol) yields four molecules of ATP. However, two ATP molecules are used up in the production of glucose-6-phosphate from glucose and fructose-1, 6- disphosphate from fructose-6-phosphate. The net production of ATP is thus only two ATP molecules per mole of glucose during anaerobic glycolysis or fermentation. 3.5.2 Fates of Pyruvate The production of pyruvate from glucose (glycolysis) occurs in a similar manner in most cells. In contrast, the fate of the pyruvate so produced varies with cellular conditions and the nature of the organism. Three common fates for pyruvate are of prime importance: conversion into acteyl CoA, lactate, and ethanol. 1. Oxidation to Acetyl CoA - Under aeriobic conditions, pyruvate is oxidized to acetyl CoA catalyzed by pyruvate dehydrogenase. 2. Formation of Lactate - Pyruvate is converted in to lactate by the action of lactate dehydrogenase in the presence of NADH + H+. NAD+ is formed. 26 | P a g e 3. Ethanol Fermentation -- Ethanol fermentation is the enzymatic anaerobic conversion of pyruvate to ethanol and carbon dioxide. The first step is conversion of pyruvate to ethanol in a decarboxylation reaction to produce acetaldehyde. Second step involves reduction of acetaldehyde to produce ethanol. A key concept in considering these fates of pyruvate is the need for a continuous supply of NAD+ for glycolysis. As glucose is oxidized to pyruvate in glycolysis, NAD+ is reduced. It is significant that each pathway of pyruvate metabolism includes provisions for regeneration of NAD+ from NADH so that glycolysis can continue. 3.5.3 Oxidation of Pyruvate to Acetyl CoA Pyruvate derived from glucose by glycolysis, is oxidatively decarboxylated to acetyl-CoA before entering into citric acid cycle. The reaction is catalysed by multiple enzyme complex pyruvate dehydrogenase, located in the mitochondria. Thiamine pyrophosphate, coenzyme A, lipoic acid and NAD+ are coenzymes needed for the reaction. The acetyl CoA is fed into citric acid cycle and oxidized to CO2 in citric acid cycle. The NADH+H+ is reoxidised to NAD+ via electron transport chain in mitochondria and during this 3 molecules of ATP are synthesized. Four different vitamins required in human nutrition are vital components of this system: thiamine (inTPP), riboflavin (in FAD), niacin (in NAD) and pantothenete (in CoA). The acetyl CoA is fed into citric acid cycle and oxidized to CO2 in citric acid cycle. The NADH+H+ is reoxidised to NAD+ via electron transport chain in mitochondria and during this 3 molecules of ATP are synthesized. CH3COCOOH+ NAD++ CoA-SH→ CH3 COS~CoA+ NADH+H++ CO2 (Pyruvate dehydrogenase) Pyruvate Acetyl CoA Chapter 6: Citric acid cycle 3.6.1 Citric acid cycle The citric acid cycle (Kreb’s cycle, Tricarboxylic acid cycle) is a series of reactions in mitochondria that bring about the catabolism of acetyl residues to CO2 and water in aerobic condition, lead to the release of most of the free energy which is captured as ATP. The acetyl residues are in the form of acety1- CoA (CH3-CO~S-CoA, active acetate), an ester of coenzyme A. Coenzyme A contains the vitamin pantothenic acid. The major function of the citric acid is to act as the final common pathway for the oxidation of carbohydrate, lipids, and protein.This is because glucose, fatty acids, and many amino acids are all metabolized to acetyl-CoA or intermediates of the cycle. It also plays a major role in gluconeogenesis, transamination, deamination, and lipogenesis. Several of these processes are carried out in many tissues but the liver is the only tissue in which all occur to a significant extent. Reactions of the citric acid cycle liberate reducing equivalents and CO2 in eight steps. 27 | P a g e 3.6.1.1 Step 1: Condensation of Acety1-CoA with oxaloacetate to form Citrate The initial condensation of acety1- CoA with oxaloacetate to form citrate is catalyzed by condensing enzyme, citrate synthase, which effects synthesis of a carbon to carbon bond between the methyl carbon of acety1-CoA and the carbony1 carbon of oxaloacetate. Acety1- CoA + Oxaloacetate ↔ Citrate (Citrate synthase) 3.6.1.2 Step 2: Conversion of Citrate to Isocitrate via Cis-Aconitate Citrate is converted to isocitrate by the enzyme aconitase which contains iron in the Fe2+ state in the form of an iron- sulfur protein (Fe:S). This conversion takes place in two steps: dehydration to cis- aconitate, some of which remains bound to the enzyme, and rehydration to iocitrate. Citrate ↔ cis-aconitate ↔ isocitrate (Aconitase for both reactions) 3.6.1.3 Step 3: Dehydrogenation of Isocitrate to Oxalosuccinate and then to α-ketoglutarate Isocitrate undergoes dehydrogenation in the presence of isocitrate dehydrogenase to form oxalosuccinate.The linked oxidation of isocitrate proceeds almost completely through the NAD+ dependent enzyme isocitrate dehydrogenase. There follows decarboxylation of oxalosuccinate to α- ketoglutarate, also catalyzed by isocitrate dehydrogenase. A CO2 molecule is liberated. Mn2+(or Mg2+) is an important component of the decarboxylation. Isocitrate + NAD+ ↔ Oxalosuccinate + NADH+ H+ (Isocitrate dehydrogenase) Oxalosuccinate ↔α-ketoglutarate + CO2 3.6.1.4 Step 4: Decarboxylation of α-ketoglutarate to succiny1-CoA α-ketoglutarate undergoes oxidative decarboxylation. The reaction is catalyzed by a α-ketoglutarate dehydrogenase complex, which requires cofactors thiamin pyrophosphote, lipoate, NAD, FAD and CoA resulting in the formation of succinyl-CoA, a high- energy thioester and NADH. Arsenic inhibits the reaction, causing the substrate α-ketoglutarate to accumulate. 3.6.1.5 Step 5: Conversion of Succinyl CoA to Succinate Succinyl-CoA is converted to succinate by the enzyme succinate thiokinase (succinyl CoA synthetase). High-energy phosphate (ADP+Pi →ATP) is synthesized at the substrate level because the release of free energy from the oxidative decarboxylation of α- ketoglutarate. The reaction requires GDP or IDP which is converted to GTP or ITP in the presence of inorganic phosphate which is then converted to ADP to ATP. 3.6.1.6 Step 6: Dehydrogenation of Succinate to Fumarate Succinate is metabolized further by undergoing a dehydrogenation catalyzed by succinate dehydrogenase.It is the only dehydrogenation in the citric acid cycle that involves the direct transfer of hydrogen from the substrate to a flavorprotein without the participation of NAD+. The enzyme contains FAD and iron-sulfur (Fe:S) protein. Fumarate is formed. 3.6.1.7 Step 7: Addition of water to Fumarate to give Malate Fumarase (fumarate hydratase) catalyzes the addition of water to fumarate to give malate. In addition to being specific for the L-isomer of malate, fumarase catalyzes the addition of the elements of water to the double bond of fumarate in the tans configuration. 28 | P a g e 3.6.1.8 Step 8: Dehydrogenation of Malate to Form Oxaloacetate Malate is converted to oxaloacetate by dehydrogenation catalysed by the enzyme malate dehydrogenase, a reaction requiring NAD+. One turn around the citric acid cycle is completed. An acetyl group, containing two carbon atoms, is fed into the cycle by combining it with oxaloacetate. At the end of the cycle a molecule of oxaloacetate was generated. The enzymes of the citric acid cycle, except for the ∝ - 3.6.2 Oxidative phosphorylation ketoglutarate and succinate dehydrogenase, are also found outside the mitochondria. As a result of oxidations catalyzed by dehydrogenase enzymes of the citric acid cycle, three molecules of NADH and one molecule of FADH 2 are produced for each molecule of acety1-CoA catabolized in one revolution of the cycle. These reducing equivalents are transferred to the respiratory chain in the inner mitochondrial membrane. Pyruvate Isocitrate ADP+Pi→ATP ADP+Pi→ATP ADP+Pi→ATP ↑ ↑ ↑ Αketoglutarate → NAD → FMN → Co Q → Cytb → CytC1 → CytC→ Cyta → Cyta3 Malate 29 | P a g e  During passage along the respiratory chain, reducing equivalents from each NADH generate three high- energy phosphate bonds by the esterification of ADP to ATP in the process of oxidative phosphorylation. However, FADH 2 produces only two high- energy phosphate bonds because it transfers its reducing power to Co Q, by passing the first site for oxidative phosphorylation in the respiratory chain. A further high-energy phosphate is generated at the level of the cycle itself (i.e., at substrate level) during the conversion of succiny1 -CoA to succinate. Thus, 12 ATP molecules are generated for each turn of the cycle ATP Production from glucose One molecule of glucose is converted to 2 molecules of pyruvate by glycolysis and the pyruvate is further converted to acetyl CoA by pyruvate dehydrogenase before entering into citric acid cycle. During this process two more molecules of NADH are available for oxidation by the electron transport cycle reoxidation route to yield 6 ATP molecules. During the conversion of glucose to pyruvate two more molecules of ATP are available through substrate-linked phosphorylation. Thus the total number of ATP produced by the aerobic oxidation of glucose to carbon dioxide and water is 38(12+3=15x2 30+8=38) ATP molecule per each glucose molecule. Citric acid cycle intermediates are used for other metabolic purposes.  It is an amphibolic pathway.  It functions not only in the oxidative degradation of carbohydrates, fatty acids and amino acids but also as a source of precursors for other metabolic pathways. ATP production in mitochondria Name of enzyme Reaction Catalyzed ATP molecules formed Isocitrate dehydrogenase Respiratory chain oxidation of NADH +H+ 3 α-Ketoglutrate dehydrogenase Respiratory chain oxidation of NADH +H+3 3 Succinate thiokinase Phosphorylation at substrate level 1 Succinate dehydrogenase Respiratory chain oxidation of FADH2 2 Malate dehydrogenase Respiratory chain oxidation of NADH +H+ 3 Net 12 3.6.3 Significance of Citric acid cycle It acts as a common pathway for the oxidation of carbohydrate, lipids and proteins because glucose, fatty acids and many aminoacids are metabolised to acetyl-CoA which is finally oxidises in the citric acid cycle.The reducing equivalents in the form of hydrogen and electrons are formed by the action of specific dehydrogenation during the oxidation of acetyl CoA in the cycle. These reducing equivalents then enter the respiratory chain when large amounts of high energy phosphate are generated by the oxidative phosphorylations. The enzymes of citric acid cycle are located in the mitochontrial matrix either or attached with inner mitochondrial membrane which facilitates the transfer of reducing equivalents to the adjacent enzymes of electron transport chain which is situated in the inner mitochandrival membrane. The citric acid cycle is amphibolis(dual) in nature. 30 | P a g e 3.6.4 Mitochodria and ATP Production The mitochondrion is called the power house of the cell, since it is within this organelle that most of the capture of energy derived from oxidation of food. The system in mitochondria that couples oxidation to the generation of the high energy intermediate, ATP, is termed oxidative phosphorylation. Mitochondria have an outer membrane that is permeable to most metabolites, an inner membrane which is selectively permeable and which forms folds or cristae, and a matrix with the inner membrane. They are consumed together in hundreds of grams per day, depending upon body weight, age and sex.The soluble enzymes of the citric acid cycle and the enzymes of beta oxidation of fatty acids are found in the matrix, necessitating mechanisms for transporting metabolites and nucleotides across the inner membrane. All the useful energy liberated during the oxidation of carbohydrates, fatty acids and amino acids are made available within mitochondria as reducing equivalents –H or electrons. The mitochondria contain a series of catalysts called respiratory chain or electron transport particles. That collect and transport reducing equivalents and direct them to their final reaction with hydrogen to form water and the machinery for trapping the liberated free energy as high energy. 3.6.5 Electron Transport Chain The electron transport chain is a series of biochemical reactions in which electrons and hydrogen ions from NADH and FADH2 are passed to intermediate carriers and then ultimately react with molecular oxygen to produce water. NADH and FADH2 are oxidized in this process. NADH + H+ → NAD+ + 2H+ +2e- FADH2 → FAD + 2H+ +2e- The major components of the respiratory chain are as follows. NAD → FAD→CoQ→ Cyt b → Cytc1 →Cyt c → Cyt a→ Cyt a3 →O2 The enzymes and electron carriers needed for the ETC are located along the inner mitochondrial membrane. Within this membrane are four distinct protein complexes, each containing some of the molecules needed for the ETC process to occur. These four protein complexes, which are tightly bound to the membrane, are Complex I : NADH-coenzyme Q reductase Complex II: Succinate-coenzyme Q reductase Complex III: Coenzyme Q-cytochrome c reductase Complex iv: Cytochrome c oxidase Hydrogen and electrons flow through the chain in steps from the more electronegative components to more electropositive oxygen through a redox span of 1.1V from NAD/NADH to O2/2H2O. O2 + 4e- + 4H+ → 2H2O Energy is released at three places as the hydrogen ions and electrons flow through the respiratory chain. They are between NAD and FAD, between FAD and CoQ and between Cytochrome ‘a’ and ‘a3’. Thus 3ADP molecule captures this energy in the form of high energy phosphate and forms 3ATP. The resulting ATP passes on this free energy to drive those processes that require energy. Hence the ATP is called “the energy currency” of the cell. The production of ATP by oxidation is termed oxidative phosphorylation. 31 | P a g e Unit 4: Amino acids Chapter 1: Structure and Classification of Amino acid 4.1.1 Amino acids Amino acids are the basic structural units of proteins. Most proteins contain, in varying proportions, of the same 20 L α-amino acids. The kinds of amino acids, the order in which they are joined together, and their mutual spatial relationship dictate the three - dimensional structures and biologic properties of simpleproteins. In addition to their roles in protein, L-amino acids and their derivatives participate in intracellular functions such as nerve transmission, cell growth, and in the biosynthesis of enzymes, porphyrins, purines, pyrimidines, and urea. L-a amino acids are present in polypeptide antibiotics synthesized by microorganisms. Some amino acids occur in free or combined states and fulfill important roles in metabolic processes. For example, citrulline, and argninousuccinate participate in the formation of urea; tyrosine in the formation of thyroid hormones, and glutamate in neurotransmitter biosynthesis. About 20 - D-amino acids occur in nature. These include the D-alanine and D-glutamate of certain bacterial cell walls and a variety of D- amino acids in antibiotics. 4.1.2 Structure of Amino acids Amino acids consist of a central carbon atom chemically bonded to one hydrogen atom (H), one carboxylic acid group (-COOH) and one side chain 'R'. The side chain gives each amino acid its identity unique to each amino acid. It varies from one hydrogen atom in glycine to complex ring of carbon and hydrogen in phenylalanine. In ∝-amino acids, both the amino and carboxyl groups are attached to the same carbon atom (∝-carbon). All the amino acids except glycine have an asymmetric carbon atom (which four different groups are attached. The asymmetric carbon is a chiral center. Amino acids are classified into (a) hydrophilic or (b) hydrophobic 4.1.3 Classification of aminoacids according to the polarity of their 'R' groups. They are also classified on the basis of chemical nature of their are groups. 4.1.3.1 Based on Hydrophobic or Hydrophilic nature The amino acids in proteins may be classified into two broad groups on the basis of whether the R groups attached to the α- carbon atoms are polar (hydrophilic) or nonpolar (hydrophobic). Sl. No. Hydrophobic Sl. No. Hydrophilic Uncharged 1 Alanine 1 Asparagine 2 Isoleucine 2 Cysteine 32 | P a g e  3 Leucine 3 Glycine Nonpolar 4 Methionine 4 Glutamine 5 Phenylalanine 5 Serine 6 Proline(lminoacid) 6 Threonine 7 Tryptophan 7 Tyrosine 8 Valine Charged-Acidic 1 Glutamic acid 2 Aspartic acid Basic 1 Arginine 2 Histidine 3 Lysine 4.1.3.2 Based on the groups present in the side chain Based on the groups present in the side chain ‘R’ amino acids are classified as follows i) Amino acids with aliphatic groups in the side chain ii) Amino acids with sulphur-containing group in the side chain iii) Amino acids with carboxylic group in the side chain iv) Amino acids with basic group in the side chain v) Amino acids with heterocyclic group in the side chain vi) Amino acids with aromatic group in the side chain 4.1.3.2.1 Amino acids with aliphatic groups in the side chain 33 | P a g e 4.1.3.2.2 Amino acids with sulphur-containing group in the side chain 4.1.3.2.3 Amino acids with carboxylic group in the side chain 4.1.3.2.4 Amino acids with basic group in the side chain 4.1.3.2.5. Amino acids with aromatic group 4.1.3.2.6 Amino acids with heterocyclic in the side chain group in the side chain 34 | P a g e Chapter 2: Properties, Reactions and Function of Amino acids 4.2.1 Properties and reactions and functions of aminoacid Amino acids exhibit different properties a. Physical properties d. Acid base properties b. Stereoisomerism e. Charges c. Optical activity f. Reaction with Ultra violet light 1.Amino acids are soluble in polar solvents such as water and ethanol 4.2.1 Physical properties 2.They are insoluble in non polar solvents such as benzene and ether. 3.Their melting point is above 200°C 4.2.2. Stereoisomerism Compounds with chiral center exhibit stereoisomerism and occur in two different forms. The stereoisomer of all chiral compounds having a configuration related to that of L glyceraldehydes are designated “L” and those related to D glyceraldehydes are designated “D". The amino acids present in protein molecules are the L stereoisomer. D amino acids are also found in some natural peptides. 4.2.3 Optical activity The tetrahedral orientation of four different groups about the ∝ carbon atom confers optical activity on amino acids. It is the ability to rotate the plane of plane-polarized light. Except glycine, each amino acids has at least one asymmetric carbon atom and hecnce is optically active. Some amino acids found in proteins are dextrorotatory (+) and some levorotatory (-) at pH 7.0 4.2.4 Acid-base properties of amino acids Amino acids contain a carboxyl group (acidic) and an amino group (basic) and hence they behave as acids and bases, that is, they are ampholytes. Glycine, the simplest of all amino acids, can exist in three different ionized states, depending upon the pH of the solution. At around neutral pH, both the α-amino and α-carboxyl groups are ionized. And the molecule is a dipolar or “Zwitterion”. The pH at which the dipolar ion is electrically neutral is called the isoelectric point (pI ) and amino acids have no net charge and do not move in an electrical field. 35 | P a g e 4.2.5 Charges of amino acids Amino acids may have positive, negative, or zero net charge. Amino acids bear at least two ionizable weak acid groups, a -COOH and an -NH3+. In solution, two forms of these groups, one charged and one un-charged, exist in protonic equilibrium: R-COOH ↔ R-COO- + H+ R-NH3+ ↔ R-NH2+H+ R-COOH and R-NH3+ are the protonated, or acidic partners. The R-COO- and R-NH2 are the conjugate bases (proton acceptors). Although both R-COOH and R-NH3+ are weak acids, R-COOH is a far stronger acid than is R-NH3+. At the pH of blood plasma or the intracellular space(pH 7.4), carboxyl groups exist almost entirely as carboxylate ions, R-COO-. Most amino groups are predominantly in the protonated form, R-NH3+. In acid medium: H++ NH3+RCOO– ↔ NH3+RCOOH In Alkaline medium: In alkaline medium the amino acid acts as acids, yielding an anion, and move towards anode in an electrical field. NH3+RCOO– + H2O ↔ NH2RCOO– + -OH On the basis of these opposite reactions depending upon the acidity and alkalinity of the solution, the amino acids are called ampholyte. Isoelectric pH (pI) of an amino acid is the pH at which it has no net charge and hence does not move in an electric feld. The ion at pI carries both + and – charges and is called ‘Zwitterion’. It is an ampholyte as it has both a proton donor and proton acceptor. In an acid medium the amino acid acts as bases, yielding cation and move towards anode in an electrical field. 4.2.6 Absorption of ultraviolet light Aromatic amino acids such as phenylalanine, tyrosine and tryptophan absorb in the UV range of the spectrum with absorption maxima at 200-230nm and 250-290 nm. Dissociation of the phenolic -OH group of tyrosine shifts the absorption curve by about 20nm towards longer wavelength. Absorption readings at 280nm are used for the quantitative determination of proteins and peptides. Histidine, cysteine and methionine absorb between 200 and 210 nm. 4.2.3 Reactions of amino acids A variety of colour reactions occur that are specific for particular functional groups in amino acids. The following are the important reactions which are useful in both qualitative and quantitative identification of amino acids 1. Reaction with Ninhydrin 2. Reaction with Sanger’s reagent 3. Reaction of amino acids with formaldehyde 36 | P a g e 4.2.3.1 Reaction with Ninhydrin Ninhydrin oxidatively decarboxylates amino acids to CO2, NH3, and an aldehyde with one less carbon atom than the parent amino acid. The reduced compound hydrindantin reacts with another molecule of ninhydrin and the liberated ammonia, to form a purple complex (Ruhemann’s purple) that maximally absorbs light of wavelength 570 nm. This blue color forms the basis of a amino acids that can detect as little as 1 g of aminoacids. Amines, other than amino acids also react with ninhydrin, forming a blue color, but without releasing CO2. The evolution of CO2 thus indicates an ∝ amino acid. NH3 and peptides also react, but more slowly than ∝ amino acids. Proline and 4-hydroxy proline produce a yellow color with ninhydrin. 4.2.3.2 Reaction with Sanger’s reagent A very important reaction of the amino groups of amino acids is with 1- fluoro -2,3 - dinitrobenzene, FDNB, also called “Sanger’s reagent”. The reagent condenses with free amino groups in the cold in mildly alkaline solution (bicarbonate) to form dinitrophenyl(DNP) amino acid which is a fluorescing compound which is used for analysis of amino acids by chromatography. 4.2.3.3 Reaction of amino acids with formaldehyde (Sorensen’s formal titration) The carboxyl group of α -amino acids cannot be accurately titrated in water solution, because it reacts with the basic amino group to form zwitterions that are not decomposed completely at the end of alkaline indicators (phenolphthalein, thymolphtahalein). But when formaldehyde is added to the solution of an amino acid it binds to the amino group as dimethylol and the amino acid can be titrated and estimated as an acid using the above indicators. 37 | P a g e 4.2.4 Functions of Amino Acid Amino acids occupy a central position is cellular metabolism since all biochemical reaction are catalysed by enzymes composed of amino acids. Amino acids are essential for carbohydrate and lipid metabolism, for synthesis of tissue proteins and many important compounds-Adrenalin, thyroxin, melanin, histamine, porphyrins- haemoglobin, pyrimidines and purines nucleic acids, choline, folic acid and nicotinic acid, vitamins and taurine and as a metabolic source of energy or fuel. 4.2.4 Essential and Nonessential Amino Acids For nutritional purposes, amino Essential amino acids(EAA) Non-essential amino acids acids may be divided into two (NEAA) groups, essential amino acids (EAA), and the non-essential Methionine Glycine amino acids (NEAA). The essential amino acids are that, Tryptophan Cysteine which cannot be synthesized within the animal body or Threonine Alanine synthesised in sufficient amounts to meet the Valine Serine physiological needs of the Isoleucine Proline growing animal, therefore must be supplied in the diet.There are Ieucine Tyrosine 10 essential amino acids (indispensible). The remaining Phenylalanine Aspartic acid 10 are non-essential amino acids (dispensible) as they can be Lysine Asparagine synthesized in animals from other compounds. A complete Arginine Glutamic acid protein is a protein that contains Histidine Glutamine all the essential amino acids in approximately the same relative amounts in which the human body needs them. Most animal proteins, including case in from milk and proteins found in meat, fish, eggs, are complete proteins. Proteins from plants(vegetables, grains and legumes) have quite diverse amino acid patterns and some tend to be limited in one or more essential amino acids. In cereals, lysine is the limiting amino acid while in pulses it is methionine. 4.2.5 Amino acids and peptides A peptide consists of two or more amino acids linked by a peptide bond formed between the carboxyl (-COOH) group of one amino acid and the amino (-NH2) group of another amino acid releasing one molecule of water. Two amino acids are thus linked to form a dipeptide. 38 | P a g e If three amino acids are thus linked, it is a tripeptide and if many amino acids are liked they are called polypeptide. The term oligopeptide refers to a chain of 4 to 10 amino acids where as a polypeptide containing more th