Final Biochemistry 2024-2025-1 PDF

Mansoura National University Faculty of Nursing BIOCHEMISTRY FIRST LEVEL FIRST SEMSETER ACADEMIC YEAR 2024-2025  CONTENT Subject Page 1- Carbohydrate’s chemistry 2 2- Lipids...

Mansoura National University Faculty of Nursing BIOCHEMISTRY FIRST LEVEL FIRST SEMSETER ACADEMIC YEAR 2024-2025  CONTENT Subject Page 1- Carbohydrate’s chemistry 2 2- Lipids 11 3- Proteins 26 4- Enzymes 38 5- Vitamins 43 6- Minerals 54 7-Endocrines & Hormones 59 8-Regulatory mechanisms of metabolic pathway 67 References 82 Biochemistry first year First term Definition : Carbohydrates are polyhydroxyaldehydes or polyhydroxyketones or compounds yielding them on hydrolysis.They are organic compounds containing C, H & O. Importance : 1. Major source of energy, as they provide the body with about 50% of its energy. 2. Pentoses e.g. ribose and deoxyribose enter in the structure of nucleic acids (RNA & DNA). 3. Cellulose gives the bulk of stools and prevents constipation. 4. Mucopolysaccharides are important components of connective tissues. 5. Heparin is an important natural anticoagulant. Classification : 1. Monosaccharides or simple sugars: They are the simplest units of carbohydrates. They cannot give simpler forms on hydrolysis. 2. Oligosaccharides: They are sugars composed of 2:10 monosaccharide residues united together by glycosidic linkages (HC-O-CH). If 2 monosaccharides united together, we get a disaccharide. 3. Polysaccharides: They are carbohydrate polymers composed of more than 10 monosaccharide residues, united together by glycosidic linkages. They are formed by polymerization of a number of molecules of simple sugars to form one molecule. -1- Biochemistry first year First term Monosaccharides Classified according to: 1. The number of carbon atoms. 2. Wether they are aldehydes or ketones (aldoses or Ketoses (. Examples of monosaccharides: 1- Trioses (3 carbon atoms) e.g. Glyceraldehyde (aldose). Dihydroxy acetone (ketose). 2- Tetroses (4 carbon atoms) e.g. Erythrose. Erythrulose. 3- Pentoses (5 carbon atoms) e.g. Ribose, Xylose. Ribulose, Xylulose 4- Hexoses (6 carbon atoms) e.g. Glucose, Galactose, Mannose. Fructose. Trioses: H C O CH2OH H C OH C O CH2OH CH2OH Glyceraldehyd Dihydroxy e acetone Tetroses: CHO H C OH H C OH CH2OH Erythros Pentoses: e CHO CHO CHO H C H H C OH H C OH H C OH HO C H H C OH H C OH H C OH H C OH CH2OH CH2OH CH2OH D-Ribose 2-Deoxyribose D-Xylose The ketopentoses, ribulose and xylulose are intermediates in metabolism. -2- Biochemistry first year First term Hexoses: CHO CHO CHO CH2OH H C OH H C OH HO C H C O HO C H HO C H HO C H HO C H H C OH HO C H H C OH H C OH H C OH H C OH H C OH H C OH CH2OH CH2OH CH2OH CH2OH D-Glucose D-Galactose D-Mannose D-Fructose (Aldose) (Aldose) (Aldose) (Ketose) Physical properties of monosaccharides: 1. Soluble in water 2. Cyclic structure - In natural state, aldehyde & ketone groups exist in a condensed f orm as “hemiacetal” by combination with one of the alcoholic hydroxyl group. - In aldohexoses, the cyclic forms results from condensation between the aldehyde group on C 1 and the OH group of C 5 or C 4. If condensation occurs with OH group of C 5 , we get a six-membered ring, forming a pyran ring, so the sugar described as pyranose. - If condensation occurs with OH group of C 4 , we get a five membered ring forming a furan ring, so the sugar is described as furanose. H OH H OH C C H C OH H C OH HO C H O HO C H O H C H C OH O O H C OH H C Pyran Furan CH2OH CH2OH Glucopyranos Glucofuranos e CH2OH e CH2OH O H C OH O OH OH OH OH OH OH OH α-D-Glucopyranose -3- α-D-Glucofuranose  Biochemistry first year First term Haworth suggested the ring structure like a ring. In the Haworth formula, the groups written above the ring correspond to the groups written to the left in the straight chain structure, and vice versa. In aldohexoses, C 1 is is the anomeric C atom, in case of ketoses, the anomeric C atom is C 2. According to the position of-OH in C 1 in aldoses or C 2 in ketoses, there are two forms (anomers): - form: in which -OH is to the right. - form: in which -OH is to the left. HO H H OH CH2OH C C H C OH H C OH O HO C H HO C H O O OH H C OH H C OH OH OH H C H C CH2OH CH2OH OH α-D-glucose α-D-glucopyranose β-D-glucose Haworth formula CH2OH O CH2OH HO CH2 O OH CH2OH HO C OH C OH HO C H HO CH2OH HO C H H C OH O OH OH H C OH O H C OH H C OH OH CH2OH H2C α-D-fructopyranose α-D-fructofuranose 3.Configuration: - When the OH group attached to the C atom which is next to the last CH 2 OH group (penultimate C atom) is on the right, the sugar is described as D-sugar. - If the OH group is on the left, it is described as L-sugar. CHO CHO HO C H H C OH CH2OH CH2OH L-Suger (L-Glyceraldehyde) D-Suger (D-Glyceraldehyde) -4- Biochemistry first year First term Enantiomers: They are “mirror images” of each other. They can rotate plane polarized light to the same extent but in opposite direction. The D-form of sugars are majority forms utilised in the body. The L-form is exerted in urine. Steriosomers : They are compounds that have the same structural formula but differ in the arrangement of groups (spatial configuration(. - The presence of assymetric C atom allows the formation of isomers. Oligosaccharides Formed of 2-10 sugar units. According to the number of sugar uni ts, oligosaccharides are classified into disaccharides, trisaccharides, etc ….. 1. Disaccharides: Contain 2 monosaccharide units. They are classified into : A- Reducing disaccharides: as maltose, lactose and cellobiose. Maltose (Malt sugar): CH2OH CH2OH O O OH OH OH O OH OH OH - It is formed of 2 molecules of -glucose united together by -1,4- glycosidic linkage. - It is a reducing sugar due to the presence of one free aldehyde group , gives characteristic osazone. - It can be hydrolyzed by: 1- acid, 2- maltose enzyme. - It is a sweet sugar, very soluble in water. -5- Biochemistry first year First term Lactose (Milk sugar(: CH2OH CH2OH OH O O OH O OH H OH OH OH - It is formed of one molecule of -glucose and another molecule of - galactose united together by -1,4 glycosidic linkage. - It is a reducing sugar, gives osazone which is sun shaped. - It can be hydrolyzed by: 1- acid, 2- lactase enzyme. - It is not fermented by backer’s yeast. B- Non reducing disaccharides : Sucrose (Cane sugar): - It is formed by condensation of one molecule of -glucose with one molecule of -fructose through 1,2-glycosidic linkage. - It is non reducing, very sweety, very soluble, not form osazone. - It is the ordinary sugar used in diet, it occurs free in most fruits and vegetables. - Sucrose is dextro-rotatory, and on hydrolysis gives a levo-rotatory mixture of equal amounts of glucose and fructose. This mixture is called Invert sugar. This is because the levo-rotation of fructose is more than the dextro-rotation of glucose. The enzyme responsible for this inversion of rotation from dextro to levo is called invertase, and is found in yeast. - Hydrolysis of sucrose also occurs by sucrase enzyme. 2. Trisaccharides: contain 3 monosaccharide units e.g. Raffinose = galactose + glucose + fructose. 3. Tetrasaccharides: contain four monosacoharide units e.g. Stachyose = 2 galactose + glucose + fructose. -6- Biochemistry first year First term Polysaccharides They are complex substances composed of more than 10 monosaccharide units. They are classified into: 1. Homogenous polysaccharides (Homopolysaccharides): On hydrolysis they yield the same type of monosaccharide units. 2. Heterogenous polysaccharides (Heteropolysaccharides): On hydrolysis they give monosaccharides and other compounds. A- Homogenous polysaccharides : Starch: - It is the storage form of carbohydratyes in plants. - It is the main carbohydrates in our diet e.g. cereals, potatoes. - It is non reducing as there is no free aldehyde or ketone. - It is composed of -glucose units linked by 2 types of linkages -1,4 and -1,6 glycosidic linkages. - The starch granule consists of 2 components, amylose (inner part) and amylopectin (outer part). - Amylose: Forms less than 25% of the starch granule straight chain polymers, formed from -glucose units combined together by -1,4 glycosidic linkage. - Amylopectin: Forms about 75% of starch granule, branched polymer 24 to 30 glucose residues combined together by 1,4 glycosidic linkage, but at the points of branches, the linkage is α-1,6 glycosidic. - Starch on acid hydrolysis gives D-glucose units while enzymatic hydrolysis with amylase enzyme gives maltose: StarchAmylodextrinErythrodextrinAchrodextrinMaltose. - The degradation products of starch can be detected by the different colours they give with iodine: Strach Blue colour Amylase Iodine Maltose + Amylodextrin Iodine Violet colour Amylase Maltose + Erthrodextrin Iodine Red colour Amylase Maltose + Achrodextrin Iodine Colourless Amylase Maltose -7- Biochemistry first year First term Dextrins: These are partial hydrolytic products of starch. They include: amylodextrins, erythrodextrins and achrodextrins. They are used in infant feeding, as adhesives and binders. Dextrans: These are polysaccharides produced by certain bacteria when allowed to grow on sugar media. They differ from dextrins in that they are formed from -glucose units linked tgether by 1,6-; 1,4 and 1,3-glycosidic linkages (highly branched). Due to their high viscosity, low osmotic pressure and slow disintegration, they are used as plasma substitute in case of haemorrhage to increase the volume of circulating blood. Glycogen: - The storage form of carbohydrates in animals mainly in liver and muscles. - It is a branched chain polymer with 8-12 glucose units for chain. Its structure is similar to that of amylopectin of starch, i.e. formed of glucose units linked by 1,4-glycosidic linkages and 1,6-bonds at the branching points, but it contains more numerous branches than amylopectin. - On hydrolysis, it gives -D-glucose units. - It gives red colour with iodine, it is non-reducing sugar. Cellulose: - It constitutes the walls of plant cells, so it is an example of structural polysaccharide. - It is a linear polymer, composed of -glucose units linked together by - 1,4 ghycosidic linkages (No branches in cellulose). - It is not digested (as its linkage is -glucosidic linkage), so it has no nutritive value. - It is important in the intestine as it forms the bulk of stools, stimulate peristalsis and prevents constipation. It is also a medium for intestinal flora which is important for vitamin B formation. - It is insoluble and not hydrolyzed readily by dilute acids. - In partial hydrolysis with acids, cellobiose is produced, while with complete hydrolysis, -glucose is obtained. -8- Biochemistry first year First term Inulin (Fructan-Fructosan): - It is found in the tubers and roots of fruits. - Linear polymer, on hydrolysis it gives -D-fructose units. - Non digestable, and if injected I.V., it is not metabolised. - Medical uses of inulin: 1. diet for diabetics. 2. It measures the glomerular filtration rate (as it is not absorbed by the renal tubules). 3. Determination of the extracellular fluid volume (since inulin does not penetrate the cells, its dilution can be used to measure the extracellular fluid volume). B- Heterogenous (Mixed) polysaccharides: Mucopolysaccharides:(glycosaminoglycans) - They are considered as structural polysaccharides in animal tissues. Hyaluronic acid: - It is formed of N-acetylglucosamine and glucuronic acid. - It is highly viscous, involved in lubricating synovial joints. It is also found in skin, umbilical cord having protective action. N.B. The invasive power of certain bacteria is due to their content of the enzyme hyaluronidase (spreading factor), which hydrolyzes hyaluronic acid in connective tissue. Heparin: - It is formed of glucosamine, glucuronic and sulfuric acid. - It is present in mast cells. - It acts as anticoagulant, and activator for plasma lipoprotein lipase enzyme (clearing factor) which helps to clear lipemic plasma, after hydrolysis of its fat contents. Chondroitin sulphate (A, B and C): - Chondroitin sulphates A and C are formed from glucuronic acid and sulphated galactosamine. - Chondroitin sulphate B contains iduronic acid and sulphated galactosamine. - Chondroitin sulphates are found distributed in skin, tendons, cartilage and cornea having supportive functions. -9- Biochemistry first year First term Lipids are organic compounds formed mainly from alcohol (R-OH) and fatty acids (R-COOH) combined together by ester linkage (R-COOR). The main alcohol is glycerol to form simple lipid some types of fat contain phosphorus and nitrogenous base to form compound lipids. Fats are characterized by being insoluble in water and soluble in some fatty solvents for example ether, chloroform, benzene (fat solvents). R-OH + HOOC-R` R-COOR` H2O Functions of lipids in the body: 1) Lipids are used as fuel for production of energy. Lipids give more energy than carbohydrates and proteins, but the body prefers to get its energy from carbohydrates more than from fat. 2) Fats provide the body with a- Essential fatty acids (unsaturated fatty acids as linoleic, linolenic acid and arashidonic fatty acids). b- Fat-soluble vitamins (Vitamin A, D, E and K). c- Phosphorus in the form of phospholipids. 3) Fats under the skin are used as insulator protecting the body ag ainst external temperature by fixing the temperature at 37 o C. 4) Fats are used for fixation of internal organs e.g. fats around the kidney. 5) Lipids enter in the formation of cells,for example, lipoproteins which are type of fat attached with protein to form lipoprotein which enter in cell walls, muscules, mitochondria and/or microsomes. 6) Cholesterol which is member of lipids used in synthesis of hormones (adrenal cortical hormones), vitamin D 3 (Antirickets) and bile acids which is essential for solubilization of fats from gastrointestinal tract (GIT) and metabolism. 7) It provides the milk with its fat content which is essential for normal growth of neonates. - 10 - Biochemistry first year First term Glycerol CH2 OH CH OH It is a polyhydric alcohol containing 3 hydroxy groups : CH2 OH Properties: 1. It is soluble in water and alcohol. 2. It combines with fatty acids by ester linkages to form triglycerides. Uses in medicine: 1. Glycerol enters in all cosmetic preparations. 2. Nitroglycerine is used as vasodilator especially for the coronary arteries. So it is used for treatment of angina pectoris. 3. Glycerol is used for treatment of glucoma (increased intra-ocular pressure) due to its ability to dehydrate the tissue from its excess water. Fatty Acids General formula: R-COOH Types of fatty acids: 1) Saturated fatty acids. 2) Unsaturated fatty acids. Saturated fatty acids: They are called saturated because they donot contain double bonds. The most important saturated fatty acids are: - Butyric acid containing 4 carbon atoms and is present mainly in butter. CH3 –(CH2 ) 2 –COOH. - Caproic acid containing 6 carbon atoms and is present in butter and coconut oil. CH 3 -(CH 2 ) 4 –COOH. - Palmitic acid containing 16 carbon atoms and is present in both animal and vegetable fats. CH 3 -(CH 2 ) 14 –COOH. - Stearic acid containing 18 carbon atoms and is present in both animal and vegetable fats. CH 3 -(CH 2 ) 16 –COOH. - Lignoceric acid containing 24 carbon atoms and is present mainly in the brain and myelin sheaths of nerves. CH 3 -(CH 2 ) 22 –COOH. - 11 - Biochemistry first year First term Unsaturated fatty acids: They are called unsaturated due to presence of double bonds. They can be classified according to the number of double bonds into: a) Unsaturated fatty acids containing one double bond: - Palmitoleic acid (unsaturated palmitic acid) 16:1(9(. CH3 –(CH2 ) 5 –CH 10 =CH 9 –(CH2 ) 7 – COOH. - Oleic acid (unsaturated stearic acid) 18:1(9). CH3 –(CH2 ) 7 –CH 10 =CH 9 –(CH2 ) 7 –COOH. - Nervonic acid (unsaturated lignoceric acid) 24:1(15). CH3 –(CH2 ) 7 –CH 16 =CH 15 –(CH2 ) 13 –COOH. b) Unsaturated fatty acids containing more than one double bond : - Linoleic acid containing 2 double bonds 18:2(9,12). CH 3 –(CH2 ) 4 –CH 13 =CH 12 –CH 2 –CH 10 =CH 9 –(CH2 ) 7 –COOH. - Linolenic acid containing 3 double bonds 18:3(9,12,15). CH 3 –CH 2 –CH 16 =CH 15 –CH 2 –CH 13 =CH 12 –CH 2 –CH 2 =(CH2 ) 7 –COOH. - Arachidonic acid containing 4 double bonds 20:4(5,8,11,14). CH 3 –(CH 2 ) 3 –CH=CH–CH 2 –CH=CH–CH 2 –CH=CH–CH 2 –CH=CH–(CH 2 ) 4 – COOH. This type of unsaturated fatty acids (containing more than one double bond ) is characterized by : a) It is essential for normal growth. b) It is not formed in the body and so must be taken in the diet or it is essential to be taken from the outside. Accordingly they are called essential fatty acids because they must be taken in diet Classification of Lipids Simple Compound Derived Substances associated with lipids - 12 - Biochemistry first year First term I. Simple Lipids They are called simple because they are formed only from alcohol and fatty acids. They can be classified according to type of alcohol into : A) Neutral fat if the alcohol is glycerol. B) Waxes if the alcohol is higher than glycerol. A) Neutral fat: Neutral fats his is the most important and most abundant group of fats in nature. Fats Oils Fats and oils have the same chemical structure and the same chemical properties but as regards the physical properties, they differ only in their state at room temperature. Fats are solid while oils are liquid. Chemical structure of fats and oils: They are formed from glycerol and three molecules of fatty acids combined together by ester linkages. Since the three hydroxyl groups of glycerol are esterified with 3 molecules of fatty acids forming neutral fat, the neutral fat is also called triglyceride or triacylglycerol:- CH2 OH CH2 O COR1 HOOC R1 - 3H2O CH OH HOOC R2 CH O COR2 + HOOC R3 CH2 OH CH2 O COR3 glycrol Fatty acids Triacylglycrol If, the three fatty acids are one and the same type, the neutral fat is a simple glyceride e.g. tripalmitin. If the three fatty acids are different from each other, the neutral fat is a mixed glyceride. e.g. palmitostearin. CH2 O COR1 CH2 O COR1 CH2 O COR1 CH OH CH O COR2 CH O COR2 CH2 OH CH2 OH CH2 O COR3 1-Acylglycerol 1,2-Diacylglycerol Triacylglycerol - 13 - Biochemistry first year First term Rancidity: Rancidity is the unpleasant odour and taste developed after exposing fats and oils to light, oxygen, moisture and warm temperature and lipase enzyme secreted by some bacteria. Effect of rancid fat: 1. The products of rancidity are toxic. 2. Products of rancidity destroy other factors of food essential for life e.g. a- Vitamins A and C. b- Essential fatty acids. To protect the food from rancidity, the following measures can be done : 1) Keep the food in cold place to inhibit the action of lipase enzyme. 2) Prevent oxidation : This done by: a) Replace O 2 by inert gas e.g. N 2. b) Put the food in vacuum. c) Add antioxidant material to the food e.g. vitamin E. B) Waxes: These are esters of higher fatty acids combined with certain alcohols but not glycerol. Properties: 1- Insoluble in water but soluble in fat solvents. 2- Not easily hydrolysed. 3- Not digested. II. Compound Lipids They are called compound because they contain beside the alcohol and fatty acids other substances e.g. phosphate group, nitrogenous base , carbohydrate, proteins, sulphur and amino groups. Types: (l) Phospholipids. (2) Glycolipids. (3) Lipoproteins. (4) Sulpholipids. (5) Amino lipids. - 14 - Biochemistry first year First term (1) Phospholipids They are called phospholipids because they contain phosphorus. 1- Phosphatidic acid: It is formed from glycerol, 2 fatty acids and phosphate group. There is no nitrogenous base. It is the simplest type of phospholipids. CH2OCOR1 CHOCOR2 O CHO P OH OH 2- Cardiolipin: A diphosphatidyl glycerol, is a phospholipid found i n membranes of mitochondria. It is formed from 3 molecules of glycerol, 4 fatty acids and 2 phosphate groups. Cardiolipin is used as the antigen in the common serologic test for syphilis, the Venereal Disease Research Laboratory (VDRL). CH2OCOR1 CH2O P O H2C CHOCOR3 CHOCOR2 CHOH CH2OCOR4 CH2O P O CH2 3- Lecithins: (phosphatidyl choline) It is formed of glycerol, two fatty acids, phosphoric acid and choline which is a nitrogenous base. Lecithins without choline is nothing but phosphatidic acid. CH2 O COR1 CH O COR2 CH3 OH CH3 H2 H2 CH2 O P O C C N+ CH3 O OH - 15 - Biochemistry first year First term So lecithins are sometimes called phosphatidyl choline. One of the fatty acids of lecithins is saturated e.g. palmitic acid or stearic acid, while the other fatty acid is unsaturated e.g. oleic acid, linoleic acid, linolenic acid or arachidonic acid. If the phosphoryl choline group is attached to OH group of carbon number 3 of glycerol,  -lecithin is produced. If attached to OH group of carbon number 2,  -lecithin is produced. Lecithin can be hydrolysed by: a) Alkali producing soap, glycerol, phosphoric acid & choline. b) Enzyme (lecithinase A) present in venom of snakes causing removal of unsaturated CH2 O CO R CH OH CH2 O P O choline OH O Lysophosphatidylcholine fatty acid producing a compound called lysolecithin which is haemolytic in action. Importance: 1. Enter in the formation of cell walls and cell membranes. 2. Necessary for fat metabolism in liver. Sources: (1) Egg yolk (2) Brain tissue. 4- Cephalins: Their chemical structure is exactly as lecithins but they differ only in the base, which may be: a) Ethanol amine and so - called phosphatidyl ethanol amine. b) Serine and so - called phosphatidyl serine. By lecithinase A 2 , lysocephalins are produced which are haemolytic in action. CH2 O COR1 CH2 O COR1 CH O COR2 CH O COR2 H2 OH C NH3+ OH H2 H2 CH2 O O CH CH2 O P O C C NH3+ P COO- O O Phosphatidyl ethanol amine Phosphatidyl serine - 16 - Biochemistry first year First term Importance: 1) They enter in the formation of cell walls exactly as lecithins. 2)They enter in the formation of thromboplastin which is a substance necessary for blood clotting. Sources: Brain tissue. 5- Inositides: CH2 OCOR1 CHOCOR2 OH OH OH CH2 O P O O OH OH OH Some systems of classification consider them as a group of cephalins. But chemically they are a group distinct from cephalins. They are similar to lecithin or cephalin but inositol replaces the base. Some types of inositides contain an additional phosphate group or groups(1-3) attached to inositol by ester linkage. These are called phospho - inositides. Sources: Brain tissue. 6-Plasmalogens : CH2 O CH CH R1 CHOCOR2 OH CH2O P O CH2CH2NH2 O They are a special class of phosphoglyceride in which the fatty acid on the carbon atom number one is replaced by an  -  unsaturated fatty alcohol, forming ether linkage.The second carbon atom of glycrol is attached with other fatty acid by ester linkage. Also,they are formed from the combination of glycerol, phosphoric acid and a base (choline, ethanol amine or serine). Sources: Brain tissue, muscles, liver and eggs. - 17 - Biochemistry first year First term 7- Sphingomyelins: In this type of phospholipids there is no glycerol. It is formed from sphingosine base or sphingol. To the last OH group a phosphoryl choline group is attached by an ester linkage. To the amino group of sphingosine a fatty acid is attached by an amide linkage. N-acyl derivatives of sphingosine are called ceramides and have been found in the free state in the spleen, in the liver, and red cells. The fatty acid present may be stearic, lignoceric and nervonic acids (in brain and nerves) or palmitic and lignoceric acids (in spleen and lung ). Sources: Brain tissue, myelin sheaths of nerves, lung, and spleen. CH3 (CH2)12 CH CH CH OH O CH NH C R Ceramide CH2 OH Monounsaturated alchol CH3 (CH2)12 CH CH CH OH CH NH2 CH2 OH Ethanol amine Sphingosin base CH3 (CH2)12 CH CH CH OH O CH NH C R CH3 CH2 O P O CH2 CH2 N+ CH3 O O- CH3 Sphingomyelin - 18 - Biochemistry first year First term (2) Glycolipids They are present in cerebral hemispheres and so called also (cerebrosides). We have different types of cerebrosides : a) Kerasin. b) Phrenosine or cerebron c) Nervon. d) Hydroxy nervon. The four previous types of glycolipids are present in the white matter of cerebral hemispheres and in myelin sheaths of nerves,spleen and liver. Gaucher,s disease produced by increase deposition of cerebrosides in the spleen and liver. (3) Lipoproteins The serum lipoproteins are complexes of lipids and specific proteins called apoproteins. These particles are in a constant state of synthesis, degradation, and removal from the plasma. These particles include the chylomicrons (CM), very low density lipoproteins (VLDL),intermediate density lipoprotein(IDL) ,low density lipoproteins (LDL), and high density lipoproteins (HDL). Lipoproteins function both to keep lipids in a soluble state,as they transport them in the serum ;thus they provide all tissues with its requirements.Thus to provide all tisssues with its delivering content of lipids.Human easily accept a gradual deposition of lipids in tissues (especially TG and cholesterol). If this process increased than normal level,this leads to lipid deposition in blood vessels which leads to accumulation of fats (This condition known as atheroscelerosis) Plasma lipids and lipoproteins:- Lipids are used for production of energy,insoluble in water ,the problem is presented of transporting a large quantity of hydrophobic material(lipid) in an aqueous envinment(blood plasma).This is solved by associating the more insoluble lipids (TG and cholesterol) with more polar ones(phospholipids and cholesterol ester) then combining them with protein to form a hydrophilic lipoprotein complex ,thus TG de rived from intestinal absorption of fat or from liver are transport ed in the blood as chylomicrons and very low density lipoproteins(VLDL). Fat is released from adipose tissue in the form of free fatty aci ds and carried in the unesterified state in the plasma as an albumin free fatty acid complex.Thus many classes of lipids are transported in the blood as lipoproteins. - 19 - Biochemistry first year First term Pure fat is less dense than H 2 O,it follows that as the proportion of lipid to protein in the lipoprotein increases,the denisty decreases.Thus denisty is used in separating the various lipoproteins in plasma by ultracentrifugtation or electrophoresis. The denisty of lipoproteins increases as the protein content increases and the lipid content is decreases(falls)and as the size of the particles becomes smaller. In adddition to the use of techniques depending on their denisty,also lipoproteins may be separated according to their electrophoretic properities.A part from FFA ,4 major groups of lipoproteins have been identified that are important physiologicaly and in clinical diagnosis.These are chylomicrons,VLDL(or pre-B lipoproteins),LDL(or B-lipoproteins)and HDL(or α-lipoproteins).T.G is the predominant lipids in chylomicron and VLDL whereas cholesterol and phospholipids are the predominant in LDL and HDL.The protein moiety of lipoprotein is known as apolipoprotein or apoprotein,consisting nearly 60% ofsome HDL and as little of 1% of chylomicron. HDL is involved in the metabolism of chylomicron,VLDL and also cholesterol transport. Lipoprotein function both to keep lipids in a soluble state and also to transport lipids from liver to serum,thus provide all tissues with its requirement. Lipoproteins present in:- 1-Cell membrane 2-Mitochondrail membrane and muscles 3-Blood plasma(plasma lipoproteins).Most blood lipids are carried as lipoprotein complexes.The lipid fractions in such complex may be:TG,phospholipids,cholesterol or cholesterol ester,fat soluble vitamins and steroid hormones. -Elevated level of cholesterol which is presents in VLDL,IDL and LDL are associated with atheroscelerosis ,where as high level of HDL have a protective effect.Inhereted defect in lipoprotein metabolism leads to hypo or hyperlipoprotenemia. -Plasma lipoprotein synthesized and secreted by the liver and intestine. - 20 - Biochemistry first year First term Composition of serum lipoproteins:- Chylomicron VLDL LDL HDL Denisty(g/cm 3 ) ‹ 0.094 0.94-1.006 1.006-1.063 1.063-1.2 Protein % 2 10 22 50 Neutral fat (TG%) 85 60 8 3 Phospholipid % 8 15 20 30 Cholesterol % 5 15 50 17 Site of synthesis Intestinal mucosa Liver, From VLDL,liver Liver,intestineand (sources) and intestine VLDL Main lipids T.G(exogenous) T.G(endogenous) Cholesterol and its Phospholipids and ester cholesterol (4) Sulpholipids These are sulphate esters of kerasin or phernosine. The sulphate group is usually attached to OH group of C 3 of galactose. They are usually present in the brain. (5) Amino lipids These are lipids combined to substances containing amino groups. Since proteins contain amino groups, so lipoproteins can be considered as a type of amino lipids. III. Derived Lipids These are substances derived from the simple and compound lipids by hydrolysis. Derived lipid Fatty acids Hydrocarbons Alcohols Saturated Unsaturated Glycerol Higher Fatty aldehyde Vitamin D Sterols Inositol - 21 - Biochemistry first year First term IV. Substances Associated with Lipids in Nature These are substances which are always present in nature in close attachment to lipids. These substances include : 1- Vitamin A and carotenes 2- Vitamin D 3- Vitamins E or tocopherols 4- Vitamin K These will be discussed in detail in the chapter of Vitamins.These vitamins are soluble in fatty solvents only not soluble in water. 5- Steroids. Steroids: These are substances which always present in nature in close attachment with lipids. All steroids contain a ring system. The most important steroids are : 1- Sterols 2- Bile acids 3- Adrenal cortical hormones 4- Sex hormones 5- Vitamin D 6- Cardiac glycosides Sterols (solid alcohols): - There are two types of sterols:- 1-Animal sterol(cholesterol) 2-Plant sterol(ergosterol) 1) Cholesterol: Occurance in the - Adrenal cortex - Blood body:- (mainly synthesized in the liver) - Corpus luteum - Egg yolk - Brain and nerve tissue Liver and kidney Cholesterol HO - 22 - Biochemistry first year First term Importance of Cholesterol: 1. cholesterol is oxidized to give 7-dehydrocholesterol. This is stored under the skin, when exposed to UV the ring B is ruptured and the th ird bond is introduced in it to give vitamin D3: oxidation HO HO 7-dehydrocholeseterol photolysis CH2 HO Vit D3 2.Cholesterol is redulced by bacterial action of large intestine to caprosterol(i.e,removal of the double bond between 5,6. HO HO caprosterol - 23 - Biochemistry first year First term 3.The liver cholesterol can be converted to give bile acids cholic acid (3, 7, 12 trihydroxy cholanic acid), chenodeoxy cholic acid (3, 7 dihydroxy cholanic acid) and lithocholic acid (3-hydroxy cholanic acid). Cholic acid is main bile acid in the human bile. In the liver bile acids are conjugated with glycine or taurine to give glycocholic or taurocholic acid. The acids conjugated with sodium or potassium giving sodium or potassium glycocholate or taurocholate which is essential for fat digesion and absorption. OH COOH 3,7,12-Trihydroxy cholanic acid (cholic acid) HO OH 2) Ergosterol: It is a plant sterol isolated from ergot and yeast. It can give vitamin D 2 by irradiation with ultra violet light. - 24 - Biochemistry first year First term Proteins are organic compounds of large molecular weights that are made up wholly or mostly of polymers called polypeptides. Polypeptides are α- amino acids joined covalently by peptide bonds (amide linkages) between the α-carboxyl group of one amino acid and the α-amino group of another amino acid. Biological functions of Proteins: Proteins, found in all cells and in virtually all parts of cells, constitute about half of the body’s dry weight. So proteins are the most abundant and functionally diverse molecules in living systems. Virtually every life process depends on this class of molecules, for example: 1. Structural function: Elastin and keratin of hair and skin. Collagen in bone, acts like steel cables in reinforced concrete. 2. Nearly all enzymes -the body catalyst- are proteins. 3. Most hormones are proteins or polypeptides, such as ACTH, growth hormone, thyroid hormone, insulin, and glucagon. 4. Neurotransmitters, such as endorphins (31 AA') & encephalins (Penta-peptide). 5. Contractile proteins of muscles with stationary filaments, myosin, and moving filaments, actin. 6. Storage proteins (store nutrients that the organism will need), seed proteins in grains, casein of milk, ovalbumin in egg white and ferritin, the iron-storing protein in spleen. 7. Transport proteins, Hb, Mb and serum albumin. Ceruloplasmin is a copper- carrying protein. 8. Protective proteins (Defense mechanism): Antibodies (Immunoglobulins) and fibrinogen. 9. Toxins (Poisonous proteins) such as snake venom, diphtheria toxin. 10. Gene regulators that direct, control repair and construction of DNA. - 25 - Biochemistry first year First term Structure of Amino Acids: Although more than 100 different amino acids have been described in nature, only 20 of these species are commonly found as constituents of mammalian proteins. Each amino acid (except for proline) has a carboxyl group, an amino group, and a distinctive side chain (“R-group”) bonded to the α-carbon. Amino acids in solution, at neutral pH or at physiologic pH (approximately 7.4) predominantly dipolar ions (or Zwitterions) rather than unionized molecules. In the dipolar form of an amino acid, the carboxyl group is dissociate to form the negatively charged carboxyl ate ion (COO¯), and the amino group is protonated (NH 3 + ). The ionization state of an amino acid varies with pH. In acid solution (e.g., pH 1), the carboxyl group is unioniz ed (COOH) and the amino group is ionized (NH 3 + ). In alkaline Solution (e.g., pH 11), the carboxyl group is ionized (COO¯) and the amino group is unionized (NH 2 ). H+ O H+ O O + + H3N CH C OH H3N CH C O- H2N CH C O- CH3 H+ CH3 H+ CH3 pH 1 pH 6 pH 11 In proteins these carboxyl and amino groups are combined in peptide linkage and are not available for chemical reaction (except for hydrogen bonds). Thus, it is the nature of the side chains that ultimately dictates the role an amino acid will play in a protein. It is therefore useful to classify the amino acids according to the polarity of their side chains. CLASSIFICATION OF AMINO ACIDS: 1. Classification of Amino Acids According to the structure 1. Glycine (Gly) 2. Alanine (Ala) 3. Valine (Val) 4. Leucine (Leu) 5. Isoleucine (Ile) 6. Phenylalanine (Phe) 7. Tryptophan (Trp) 8. Methionine (Met) 9. Proline (pro) - 26 - Biochemistry first year First term O O O H2N CH C OH H2N CH C OH H2N CH C OH H Glycine CH3 Alanine CH CH3 Valine O O CH3 O H2N CH C OH H2N CH C OH H2N CH C OH CH2 Leucine CH CH3 Isoleucine CH2 Phenylalanine CH CH3 CH2 CH3 CH3 O O H2N CH C OH H2N CH C OH O CH2 CH2 C Tryptophan HN OH CH2 Methionine Proline S HN CH3 10. Aspargine (Asn) 11. Glutamine (Gln) 12. Cysteine (Cys) 13. Serine (Ser) 14. Threonine (Thr) 15. Tyrosine (Tyr) O O O H2N CH C OH H2N CH C OH H2N CH C OH CH2 Aspargine CH2 Glutamine CH2 Cysteine C O CH2 SH NH2 C O O NH2 O O H2N CH C OH H2N CH C OH H2N CH C OH CH2 CH2 Serine CH OH Threonine Tyrosine OH CH3 OH - 27 - Biochemistry first year First term 17 16. Aspartic acid (Asp) Glutamic acid (Glu). O O H2N CH C OH H2N CH C OH CH2 CH2 C OAspartic acid Glutamic acid CH2 OH C O OH 18. Lysine (Lys) 19. Arginine (Arg) 20. Histidine (His) O O O H2N CH C OH H2N CH C OH H2N CH C OH CH2 CH2 CH2 CH2 Lysine CH2 Arginin Histidin e CH2 CH2 e N CH2 NH NH NH2 C NH NH2 2. Classification of Amino Acids According to the Nutritional Value: A. Essential amino acids (Indispensable): They are called essential because they cannot be synthesized in the body and so they must be taken in diet. A useful statement to assist in remembering the essential amino acids is: VILLA M H = Ten Th ousand Pounds - 28 - Biochemistry first year First term These amino acids are: 1. Valine 6. Methionine 2. Isoleucine 7. Histidine 3. Leucine 8. Tryptophan 4. Lysine 9. Threonine 5. Arginine 10. Phenylalanine Some essential amino acids can be formed in the body in small quantities which are not sufficient for the physiological requirements of the body. These amino acids are also called Semi-essential or semi-indispensable amino acids e.g. arginine and histidine. B. Non essential amino acids (Dispensable): These amino acids are synthesized in the body in sufficient amounts and so it is not essential to be taken in diet. Some of these non-essential amino acids are more important than the essential amino acids e.g. glycine, glutamic acid and serine. The non essential amino acids are: 1. Glycine 6. Tyrosine 2. Alanine 7. Aspartic acid 3. Serine 8. Glutamic acid 4. Proline 9. Aspargine 5. Cysteine 10. Glutamine Functions of amino acids in Man: Unlike carbohydrates and fats, there are no large reserve stores of protein in the body. Thus, a continuous intake is required if tissue breakdown is to be avoided. Amino acids are: 1- Precursors for the synthesis of protein. 2- Source of energy under certain conditions. 3- Involved in the detoxication of drugs, chemicals and metabolic byproducts. 4- Involved as direct neurotransmitters or as precursors to neurotransmitters. 5- Precursors to several peptide hormones and thyroid hormone. 6- Precursors to histamine, NAD + , and other miscellaneous compounds of biological importance. 7- Amino acids act as buffers: Amino acids contain weakly acidic α-carboxyl groups and weakly basic α-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. - 29 - Biochemistry first year First term Post-translational modification of amino acids: Some proteins contain special amino acids that are formed by modification of a common amino acid following its incorporation into the polypeptide chain. 1- Collagen contains hydroxyproline and hydroxylysine, hydroxylated derivatives of proline and lysine. The added hydroxyl group stabilizes the collagen fiber. The biological importance of this modification is evident in scurvy, which results from insufficient hydroxylation of collagen due to absence of ascorbic acid (Vitamin C). The hydroxylysine residues can also be glycosylated. 2- A special amino acid, -carboxyglutamate, defective carboxylation of glutamate in prothrombin, a clotting protein, can lead to hemorrhage. Peptide Bond: In proteins, amino acids are joined covalently by peptide bonds that are amide linkages between the α-carboxyl group of one amino acid and the α- amino group of another. For example, glycine and alanine can form the dipeptide glycylalanine through the formation of a peptide bond. By convention the free amino-end (N-terminal) is written to the left and the free carboxyl-end (C-terminal) to the right. Therefore, all amino sequences are read from the N-terminal to the C-terminal end of the peptide. Like all amide linkages, the peptide bond neither accepts nor gives off protons over the pH range of 2 to 12, (i.e. can not be dissociated). Thus, the charged groups present in polypeptides consist solely of the N- terminal amino group , the C-terminal carboxyl group, and any ionized groups present in the side chains of the constituent amino acids. - 30 - Biochemistry first year First term When the compounds are named, all amino acid residues have the end of their names changed from -ine to -yl, except the C-terminal acid. for example, a tripeptide composed of N-terminal valine, a central glycine, and C-terminal leucine is called valylglycylleucine. Peptide bonds are not broken by normal handling or by conditions (such as heating alone or high concentrations of urea) that denature proteins. Strong acid or base at elevated temperatures is required to hydrolyze these bonds. The C-N bond in peptide linkage has partial double-bond character that makes it rigid and prevents the adjacent groups from rotating freely. Many ammo acids, usually more than a hundred, are joined by peptide bonds to form a polypeptide chain, which is an unbranched structure. An amino acid unit in a polypeptide is called a residue. A polypeptide chain consists of a regularly repeating part, called the main chain, and a variable part, comprising the distinctive side chains. The main chain is sometimes termed the backbone. Biologically Active Oligopeptides: 1- Glutathione (GSH): A tripeptide chemically named -Glutamyl- cysteinylglycine. It functions as antioxidant (The oxidized form is G- S-S-G), detoxifier and immune enhancer. Glutathione disulphide (GSH) (GSSG) 2- Oxytocin and Vasopressin: They are Nona-peptide hormones secreted from the posterior lobe of pituitary gland. Oxytocin causes contraction of the smooth muscles of uterus, so used for induction of labor (Delivery of the baby) , while Vasopressin (ADH) causes constriction of peripheral blood vessels leading to rise in blood pressure. - 31 - Biochemistry first year First term Classification of Proteins: 1. According to the general shape: Proteins can be classified into two types: A- Fibrous proteins: long thin fibres with axial ratio more than 10, insoluble in water. B- Globular proteins with axial ratio less than 10, soluble in water. A- Fibrous Proteins 1. Collagen 2. Fibrin 3. Elastin 4. Keratin 5. Myosin 6. Actin Fibrous proteins are present in hair, wool, skin, nails. Collagen is one of the most abundant of all proteins and is an important constituent of tendons, ligaments, cartilage, skin and the interstitial matrix. Collagens is composed of triple helical polypeptides all of which are wound tightly around each other to form a long, rigid fibrous molecule. B-Globular Proteins 1. Albumins 2. Globulins 3. Hemoglobin 4. Myoglobin 2. According to the chemical composition: Proteins can be classified into three types: i. Simple proteins. ii. Conjugated proteins. iii. Derived proteins. 1. Simple proteins on hydrolysis yield only amino acids. 1. Albumins 2. Globulins 3. Prolamines 4. Glutelins 5. Histones 6. Scleroproteins 7. Protamines 8. Scleroproteins (Albuminoids) 2. Conjugated proteins on hydrolysis yield in addition to amino acids a non- protein fraction (Prosthetic group). 1. Phosphoproteins containing phosphorous (Casein of milk). 2. Nucleoproteins containing nucleic acids (Nucleic acids). 3. Glycoproteins containing carbohydrates. 4. Lipoproteins containing lipids (CM, VLDL, LDL, HDL). 5. Chromoproteins containing colored pigments (Hb, Mb). 6. Metalloproteins containing metals (Hb, Mb, Ferritin). - 32 - Biochemistry first year First term 3- Derived proteins are the hydrolyses products of either simple or conjugated protein. Hemoglobin and Myoglobin Hemoglobin (Hb) is the oxygen-carrying system found in erythrocytes. It transports oxygen from the lungs to all tissues of the body, and aids in buffering blood. It is a hetero-tetramer protein, composed of 2 and 2 subunits. Each Hb molecule binds 4 oxygen molecules. Myoglobin (Mb) is a protein related to Hb and is found in muscle. It can take oxygen from Hb and acts as intracellular oxygen-transport system in muscles. It is a monomer. Each Mb molecule binds only one oxygen molecule. Myoglobin molecule A. Oxygen-carrying Proteins Hb and Mb are oxygen-carrying proteins. The oxygen-binding unit of both Hb and Mb is heme. Heme is a prosthetic group. Heme consists of a protoporphyrin bound to iron in ferrous state. Hemoglobin is built from four polypeptide chains linked non-covalently. Each chain has its own heme group with a single oxygen-binding site. The principal adult hemoglobin (Hb A) has two - and two -chains. Hemoglobin A2 (Hb A2) minor Hb in adults, has two - and two -chains. Unlike hemoglobin, myoglobin consists of a single polypeptide chain. Although myoglobin has a markedly different primary structure than Hb, the tertiary structure of its main chain closely resembles that of the - and -chains of Hb. The binding of one or more oxygen molecules to myoglobin does not influence subsequent oxygen binding to the same myoglobin molecule. Heme group of either Hb or Mb B. Binding Curves: The binding curve for Hb measures the amount of O2 bound by Hb vs. the partial pressure of O2. It is sigmoid or S-shaped, indicating that Hb is an allosteric and cooperative protein. - 33 - Biochemistry first year First term The binding of O2 to the first subunit causes conformational changes in the molecule which allows the other three O2 molecules to bind much more readily. In the physiological range of oxygen concentrations, this sigmoid binding curve is sharper than the hyperbolic binding curve (1st power) exhibited by the monomeric and non-cooperative Mb. This sharpness allows more O2 to be delivered for the same drop in oxygen tension between the lungs and other tissues. C. Regulation of O2 Binding The binding of O2 to Hb but not Mb is regulated physiologically by three negative allosteric effectors: hydrogen ions, CO2, and DPG. In the parlance of enzymologists, these effectors convert the conformation of Hb from the "relaxed" to the "tense" state. 1. Hydrogen ions: When deoxygenated Hb binds O2 in the lungs, hydrogen ions are released: Hb (H+)2 + 4O2 ↔ Hb (O2)4 + 2H+ In the tissues, metabolism releases acidic waste products (such as CO 2) causing an increase in hydrogen ion concentration decreasing the pH. By mass action in the equation above this cause more O2 to be released from Hb. Lowering pH shifts the oxygen dissociation curve of Hb to the right. In the lungs CO2 is excreted, the blood pH increases and Hb binds more O2. This overall process, called the Bohr effect, which has the result of increasing the amount of O2 delivered to the more active tissues. At the same time more protons are removed from tissues and delivered to the lungs. 2. Carbon dioxide (CO2) binds preferentially to deoxygenated Hb. It also has an allosteric effect like that produced by H+. In the tissues both H+ and carbon dioxide act additively to promote the release of oxygen. 3. 2,3-Diphosphoglycerate (DPG or BPG) also has an allosteric effect on Hb. BPG binds between the two -subunits of deoxygenated Hb (one DPG per Hb molecule) and stabilizes the molecule. DPG causes the steepest part of the oxygen binding curve to occur at an oxygen tension between that in the peripheral tissues and that in the lung. This arrangement permits the greatest amount of oxygen to be delivered to the tissues. - 34 - Biochemistry first year First term D. Hemoglobinopathies: 1. Sickle Cell Anemia [Hemoglobin S (Hb S) disease]: Sickle cell anemia is a genetic disease resulting from a single amino acid mutation of a Valine for a Glutamic acid in the -globin gene. While this has no effect on O2 binding. Hb S in the deoxygenated state precipitates and forms fibers that distort the erythrocytes, which are then destroyed leading to anemia. Although the homozygous state is detrimental to survival, heterozygote posses increased resistance to malaria. Hemoglobin S thus has net survival value in some geographic areas. A B Scanning electron monograph of normal (A) and sickle (B) red blood cells. The change of the β-globin molecule that causes this structure alteration results from a single base mutation in DNA, T to A, which results in the substitution of valine for glutamate in β-globin molecule. 2. Thalassemias: Thalassemias are hereditary Hemolytic disease in which an implance in the synthesis of globin chains. a) -Thalassemias: These are defects in which the synthesis of -globin chains is decreased or absent, tetramers of β-chains formed. b) -Thalassemias: Characterized by decreased or absence of -globin chains, -globin chains can not form stable tetramers and, therefore, precipitate, causing the premature death of cells.  and -Thalassemias lead to anemia, accumulation of synthesized globin chain and precipitation of other type methemoglobinemia (Fe++ F+++) inability to bind O2 Chocolate cyanosis. - 35 - Biochemistry first year First term Definition: Enzymes are "specific" protein catalysts that increase the rate of biological reactions without being changed themselves. They are synthesized by living cells and act either intracellularly or extracellularly, as follows: Substrate Enzyme Product Substrate is the substance that is acted upon by the enzyme. Nomenclature: - For hydrolases = name of substrate+ase e.g. urease, lipase, sucrase, etc. - For other enzymes = name of substrate + mechanism of action + ase e.g. succinic dehydrogenase, lactate dehydrogenase, etc. Composition: All enzymes are protein in nature. They are divided chemically into: 1- Simple protein enzymes: consisting only of proteins, e.g. pepsin, maltase, etc.. 2- Conjugated protein enzymes (Holoenzymes): consisting of a protein part and a non-protein part. The protein part of the enzyme is heat labile and known as apoenzyme. The non-protein part can be: A) Coenzymes (Cosubstrate): They are non-protein, thermolabile, organic part of the enzyme that is loosely attached to the protein part. It comes in full contact during the activity of th e enzyme. Coenzymes are derived from vitamins and help enzyme catalysis as follows: 1. For transfer of hydrogen:  Nicotinamide nucleotides: e.g. NAD + and NADP +.  Flavin nucleotides e.g. FAD and FMN.  Lipoic acid.  Glutathione (G-SH).  Coenzyme Q. - 36 - Biochemistry first year First term 2. For transfer of other groups:  L-Ascorbic acid.  Iron porphyrins (haem).  Vitamin K.  Thiamine pyrophosphate (TPP, Cocarboxylase).  Coenzyme-A (CoA-SH, Cotransacetylase).  Pyridoxal phosphate (cotransaminase and codecarboxylase).  Coenzyme-R from Biotin.  Tetrahydrofolic acid (FH 4 , THFA).  Cobamide from vitamin B 12.  UDPG = uridine diphosphate glucose. B) Prosthetic Group: It is an inorganic metal which is firmly attached to the protein part of the enzyme. For example, ascorbic acid oxidase contains copper, carbonic anhydrase contains zinc and cytochrome oxidase contains iron. Form of Enzymes: i) Zymogens (Proenzymes or Preenzymes): Enzymes secreted in inactive form especially proteolytic enzymes to prevent destruction of the proteins of the cells that synthesize them. Activation of zymogen takes place either by: 1- pH Changes: Pepsinogen pepsin. 2- Autocatalysis: Trypsinogen trypsin HCl trypsin. 3- Kinase: Trypsinogen Enterokinase trypsin. The mechanism of activation may involve unmasking of a polypeptide chain that is blocking or masking the active site of the apoenzyme. ii) Zymase: An enzyme that is secreted ready for action, such as salivary amylase. iii) Isoenzymes or isozymes: Enzymes that catalyze the same reaction but differ in their structure, properties and activity. Best example is lactate dehydrogenase (LDH) in plasma which by electrophoresis is separated into 5 isoenzymes. LDH molecule consists of 4 polypeptide chains; each may be H or M type. Thus the 5 isoenzymes are called LDH 1, 2, 3, 4 and 5. LDH1 (HHHH) is produced by heart, LDH5 (MMMM) is produced by muscles and liver and other isoenzymes are produced by other tissues. Also, creatine kinase (CK) occurs as three isoenzymes. Each isoenzyme is a dimer composed of two polypeptides, called B and M subunits. CK1=BB, CK2=MB and CK3=MM. - 37 - Biochemistry first year First term Properties: 1. Enzyme Specificity: Enzymes are highly specific in their reactions because they catalyze only one or a small group of related reactions. Specificity of enzymes is due to the nature and arrangement of the chemical groups at the catalytic site of the enzyme. This allows the enzyme to unite with and activate only one substrate or a small number of structurally-related substrates 2. Active sites: Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid chains that create a three dimensional surface complementary to the substrate. 3. Catalytic efficiency: Typically, each enzyme molecule is capable of transforming 100 to 1000 substrate molecules into product each second. The number of molecules of substrate converted into product per enzyme molecule per second is called the turnover number. 4. Location within the cell: Enzymes are distributed among many organelles within the cell. Some are founds in the cell-membrane (particulate), others are floating in the cytosol (soluble), and some are found in the mitochondria or in the nucleus. Mechanisms of enzyme action: The active site of enzyme unites with the substrate by many mechanisms: 1. Lock and key (rigid template) model: This rigid template model is still useful for understanding certain properties of enzymes as kinetic of simple substrate. + Enzyme Substrate Coenzyme Substrate - 38 - Biochemistry first year First term 2-Induced fit model: the substrate induces a conformational change in the active site of the enzyme to fit the substrate. + Substrate Enzyme Attachment of a substrate analog that is too bulky or too slim induces incorrect alignment. Factors Affecting Enzyme Activity: 1. Nature of the Enzyme ( e.g.Isoenzymes). Reaction velocity (Vo) 2. Substrate Concentration: The rate of an enzyme-catalyzed reaction increases proportionally with the substrate concentration; until a maximal velocity (V max ) is reached. Substrate concentration The leveling of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme. 3. Temperature: Heat activation Heat inactivation The reaction velocity Reaction velocity (Vo) increases with temperature until a peak velocity is reached. This increase is due to the increased number of molecules having sufficient energy to pass over the energy barrier and form the products of the reaction. 20 30 40 50 60 Temperature (oC) Further elevation of the temperature results in a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme. - 39 - Biochemistry first year First term 4. pH: pH at which maximal enzyme activity is achieved is different for different enzymes, and often reflects the [H + ] at which the enzyme functions in the body. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment. Alkaline + Pepsin Trypsin phosphatase The concentration of H Reaction velocity (Vo) affects reaction velocity in several ways: 1. Achievement of ionized or unionized state of active site of the enzyme and substrate required for their action. 2. Extremes of pH can lead to denaturation of the enzyme. 3 5 7 9 11 pH 5. Enzyme Concentration: The velocity of the reaction is directly proportional to the concentration of the enzyme. Further increase in the enzyme concentration will not increase the velocity of the reaction (within limit). 6. Concentration of Reaction Products: When the products of the reaction are removed as fast as they are formed, the reaction would be 100% complete. 7. Effect of Time: As the reaction proceeds, the velocity of the reaction decreases due to decreased concentration of substrate, and increased concentration of products , which tend to reverse. 8. Activators: They are inorganic ions which increase the activity of enzyme. Chloride ions activate amylase, magnesium ions activate kinases and calcium ions activate thrombokinase 9. Inhibitors: The activity of enzymes is inversely proportional to its inhibitor concentration. - 40 - Biochemistry first year First term Enzymes in clinical diagnosis: Plasma enzymes can be classified into two major groups: 1. Enzymes that are actively secreted into blood (small group of enzymes). For example, zymogens of the enzymes that involved in blood coagulation. Enzymes that are released from cells during normal cell turnover (large number of enzymes). They work intracellularly and have no physiologic use in plasma. Their level is very small in healthy individuals. The presence of elevated enzyme activity in plasma may indicate tissue damage that is accompanied by increased release of intracellular enzymes. Some enzymes show relatively high activity in only one or a few tissues. The increase in plasma levels of these enzymes reflects damage to corresponding tissue. Classification of Enzymes: I. Oxido Reductases Catalyze the oxidation-reduction reactions. II. Transferases They catalyze the transfer of a chemical group from one molecule to another. According to the transferred group, they are classified into: III. Hydrolases They catalyze the hydrolysis of their substrates by cleavage of bond in each through addition of water. According to the bond cleaved. IV- Lyases Or Desmolases (Splitting Enzymes) They catalyze the splitting of their substrates without hydrolysis, oxidation or reduction. According the group they split, lyases are classified into : V- Ligases Catalyze the binding of 2 molecules associated with the cleavage of a pyrophosphate bond, usually in ATP, as a source of energy. VI - Isomerases They are enzymes which catalyze the interconversion of two isomers VII- Coagulases They are enzymes necessary for blood coagulation as “Thrombin”: fibrinogen Thrombin fibrin. - 41 - Biochemistry first year First term Vitamins are organic compounds having certain properties: A- They are essential for normal growth and health. B- They are present in normal food but in small concentrations. C- They enter in many reactions in the body acting as catalysts (required for normal metabolism) D- They cannot be synthesized by humans, and therefore must be supplied by the diet. E- They can be classified according to their solubility into 2 main groups : I- Water Soluble Vitamins. II- Fat Soluble Vitamins. (I) Water Soluble Vitamins A- Vitamin C or Ascorbic acid. B- Vitamin B-complex includes the following members: 1- Vitamin B 1 or thiamine or aneurine. 2- Vitamin B 2 or riboflavin or lactoflavin. 3- Vitamin B 6 or pyridoxine or pyridoxal phosphate. 4- Folic acid or folinic acid. 5- Vitamin B 12 or cyanocobalamin. (A) Vitamin C or L-Ascorbic acid O C HO C O HO C H C HO C H CH2OH Distribution of Ascorbic acid: Citrus fruits, potatoes (particularly their skin), tomatoes, the green vegetables are good sources of vitamin C. Absorption: Readily absorbed from the small intestine and subcutaneous tissues. - 42 - Biochemistry first year First term Functions: It is a powerful antioxidant and help the body to fight infections because of its antiviral,antibacterial and immune boosting effect. Deficiency of Ascorbic acid: A deficiency of ascorbic acid results in Scurvy, a disease characterized by spongy gums, loose teeth, fragile blood vessels, swollen joints , loss of weight and anaemia due to haemorrhage from gum and delayed healing of wounds. Toxicity of Ascorbic acid: - No acute toxicity has been observed in man. - In rats, dehydroascorbic acid in excess doses produces permanent diabetes mellitus identical with that produced by alloxan. Requirements: Children 30 mg/day Adults 75 mg/day Pregnant females 100 mg/day Lactating females 150 mg/day These requirements must be increased in the presence of infection, wounds and fractures of bones. (B) Vitamin B-complex 1- Vit. B 1 or Thiamine or Aneurine Distribution: Pork, whole grains and legumes are the richest source of thiamine. Outer layer of seeds are particularly rich in thiamine. Thus, whole wheat bread is a good source of Vit. B 1 , whereas white bread prepared from milled grain is low in thiamine. Thiamine is also present in animal sources such as liver, eggs and milk. - 43 - Biochemistry first year First term Functions: 1- It is necessary for optimal growth of infants and children. 2- It increases the activity of acetyl choline at nerve ending by inhibiting acetyl choline esterase enzyme. Deficiency: Severe deficiency results in disease called: 1) Beri-Beri in areas where polished rice is the major components of the diet. a) Infantile Beri-Beri is characterized by tachycardia, vomiting, convulsion and if not treated, death occurs. b) Adult Beri-Beri is characterized by dry skin, irritability, disorderly thinking and progressive paralysis. In some cases, edema may occur and in this case the disease is described as wet Beri-Beri. Requirements: Children 0.4 mg/day Adult 1-1.5 mg/day 2- Vitamin B2, RiboHavin or Lactoflavin Riboflavin is present in tissues as biologically active forms which are : a) Flavin mononucleotide (FMN). b) Flavin adenino dinucleotide (FAD(. Distribution: Milk, eggs, liver and green leafy vegetables are good sources of riboflavin. Also, it is found in yeast, dry beans, peas and nuts. - 44 - Biochemistry first year First term Function: Riboflavin gives FAD which acts as a coenzyme for several enzymes catalyzing the removal of hydrogen. So FAD acts as a hydrogen carrier for certain enzymes. Deficiency: (1) In skin: Dermatitis. (2) Cornea: Vascularization. (3) In mouth: a) Cheilosis (fissuring at the corners of the mouth). b) Glossitis (the tongue appearing smooth and purplish ). c) Lips are red and shiny. Requirements: Children 0.6 mg/day Adult 1.5-1.8 mg/day Pregnancy 2 mg/day Lactation 2.5 mg/day 3- Pyridoxine or Vit. B 6 Vitamin B 6 is a collective term for pyridoxine, pyridoxal and pyridoxamine , all derivatives of pyridine, differing only in the nature of the functional group attached to the ring. CH2NH2 CH2OH HOH2C OH HOH2C OH Pyridoxamine Pyridoxine N CH3 N CH3 CHO CHO HOH2C OH O3P OH2C OH Pyridoxal Pyridoxal phosphate N CH3 N CH3 - 45 - Biochemistry first year First term Distribution: Good sources of the vitamin are wheat, corn, egg yolk, liver and muscle meats. Royal Jelly of bees is very rich in vitamin B 6. Functions: Pyridoxal phosphate acts as a coenzyme for a large number of enzymes , particularly those that catalyze reactions involving amino acids as : transamination, deamination, decarboxylation, condensation and desulfuration. Clinical indication for pyridoxine: Isoniazide (isonicotinic acid hydrazide), a drug frequently used to treat tuberculosis, can induce a B 6 deficiency by forming an inactive derivatives with pyridoxal phosphate. Dietary supplementation with B 6 is thus an adjunct to isoniazide treatment. Deficiency: (1) Impaired growth, due to disturbed amino acid metabolism. (2) Anemia due to decreased synthesis of hemoglobin. (3) Convulsions especially in children due to the decrease in concentration of -amino butyric acid in brain. (4) Pellagra may be result due to decreased formation of nicotinic acid. (5) Nausea and vomiting of early pregnancy as a result of depletion of vit. B 6 due to its excessive use in amino acid metabolism to synthesize the new proteins of the embryo. Causes of deficiency : 1- Dietary deficiencies in pyridoxine are rare but have been observed in newborn infants fed formulas low in vit. B 6. 2- Women taking oral contraceptives and in alcoholics. 3- Pregnancy, due to increased demand of the vitamin by the embryo. 4- Tuberculous patients taking high doses of isoniazide as it combines with pyridoxine forming hydrazone which inactivates the vitamin. Requirements: 2 mg/day which may be increased in high protein diet (as it is used in metabolism of amino acids). - 46 - Biochemistry first year First term 4- Folic acid, Folacin H2N N N H N N N H OH N COO- O COOH Functions: 1- Folic acid acts as a carrier of one carbon units which important for methylation reactions e.g. Adrenaline Noradrenaline. 2- Essential for red blood cells formation. Deficiency: 1- Megaloblastic anemia, caused by diminished synthesis of purines and thymidine leading to an inability of cells to make DNA. 2- Macrocytic anemia (as it is necessary for synthesis of red blood c ells). 3- Leucopenia (as it is used for synthesis of white cells). Requirements: No requirements, because it can be formed by bacteria of large intestine. Sometimes, pregnant or lactating women may require folic acid supplementation. In the case of inadequate absorption due to alcoholism, a commercial preparation of folic acid may be administered, either orally or by intramuscular injection. - 47 - Biochemistry first year First term (5) Vitamin B12, antipernicous anemia factor (Cyanocobalamine) Distribution: In milk, meat, eggs, liver, kidney and chicken. It is synthesized by intestinal bacteria. It is not present in plants. Function: The coenzymes of Vit B 12 have the following functions: 1- Synthesis of nucleic acid and nucleoprotein as it help in the action of folic acid. 2- Isomerization of methyl malonyl CoA that arises from fatty acids oxidation with odd numbers of carbon atoms. When the vitamin is deficient, abnormal fatty acid accumulates and become incorporated into cell membranes, including those of the nervous system. This may account for some of the neurologic manifestations of Vit B 12 deficiency. Also, decreased succinyl CoA which is incorporated in the synthesis of porphyrin ring used in synthesis of hemoglobin. Deficiency: Causes: 1- Lack of absorption due to decrease intrinsic factor secreted from the stomach as in stomach cancer. 2- Vegans are at risk of vit. B12 deficiency because this vitamin is present only in food of animal origin. Requirement: 0.6 – 1.2 g/day. - 48 - Biochemistry first year First term (II) Fat Soluble Vitamins They includes: (1) Vit. A (2) Vit. D (3) Vit. E (4) Vit. K (1) Vitamin A Chemistry: Vitamin A is a complex primary alcohol. It is derived from certain carotenoids which are hydrocarbon pigments (yellow to red) and widely distributed in nature. The most important carotenoids (provi tamins) are - carotene, -carotene. -Carotenes Vitamin A (Retinol) Distribution: The best sources of Vit A are Cod liver oil and other fish liver oils, the liver of other animals, butter, eggs and cheese. The provitamin occurs most abundantly in carrots and other yellow vegetables e.g., squash, sweet potatoes, tomatoes and many green vegetables particularly broccoli, sp inach and beet greens. Function: Responsible for accommodation and visual process. Deficiency: (I) In eyes: deficiency of Vit A leads to: 1. Night blindness 2. Dry eye (II) Inflammation of the skin. (III) Respiratory tract infections (IV) Urinary tract infections. Requirements: Adult: 5000 IU/day. Increase in pregnancy to 6000 IU/day. Increase in lactation to 8000 IU/day. - 49 - Biochemistry first year First term (2) Vitamin D (Cholecalciferol) Chemistry: Vitamin D is steroid in nature (sterol) and occurs mainly in animals. It is derived from 2 precursors that occur in nature: a) Ergosterol: Producing Vit D 2. b) 7-dehydro cholesterol: Producing Vit D 3. CH2 CH2 HO Ergocalciferol HO Cholecalciferol (Vit D2) (Vit D3) Occurrence: 1- Cod liver oil and other fish liver oils (halibut liver oil a nd tuna liver oil). 2- Sardines and salmon. 3- Egg yolk is rich in Vit D 3 but milk is poor in it. 4- Green plants contain small quantities and mushrooms contain slightly greater amounts. Function: 1. Increases uptake of calcium 2. Increases the absorption of phosphate. 3. Regulates calcium and phosphorus levels in blood. 4. Minimizes loss of calcium by the kidney. Deficiency: Rickets in children & Osteomalacia in adults Requirements: 400-800 IU/day for infants. 400 IU/day for adults. 800 IU/day for pregnancy. Toxicity: High doses (100,000 IU for weeks or months) can cause loss of appetite , thirst, nausea, constipation and polyuria. Enhancement of Ca ++ absorption and bone resorption leads to hypercalcaemia which can lead to deposition of calcium in many organs such as kidney and arteries. - 50 - Biochemistry first year First term (3) Vitamin K or Naphthaquinone Distribution: Vit K is found in cabbage, couli flower, spinach, egg yolk and liver. Also present in tomatoes and rice. There are extensive synthesis of the body by bacteria in the gut. O Phylloquinone H (Vit K1) 3 O O O Menaquinone (Vit K2) Menadione (n=6, 7 or 9) n H (Vit K3) O O Functions: Help blood clotting Role of liver in blood clotting: 1- Synthesis of fibrinogen, prothrombin and other clotting factors as V, VII, IX and X. 2- Synthesis of cholic acid and bile salts necessary for absorption of Vit K from the intestine. Deficiency: A true Vit K deficiency is unusual because adequate amounts are generally produced by intestinal bacteria or obtained from diet. If the bacterial population in the gut is decreased for example by antibiotics, the amount of endogenous formed vitamin is depressed and can lead to hypoprothrombinemia in the malnutritioned individuals. In addition, certain second generation cephalosporins (e.g., cefoperazone and cefamandole) cause hypoprothrombinemia, apparently by warfarin-like mechanism. This condition needs for treatment supplementation with Vit K. - 51 - Biochemistry first year First term Deficiency of Vit K in newborn : Newborn have sterile intestine and cannot initially synthesize Vit K. Because human milk provides only 1/5 of the daily requirement of Vit K, it is recommended that all newborns receive a single intramuscular dose of Vit K as prophylaxis against haemorrhagic disease. Requirements: No requirements under physiological conditions. (4) Vitamin E or Tocopherols. CH3 H3C O HO CH3 α-Tocopherol Distribution: (I) Plants: 1. Mainly in green plants as lettuce. 2. Vegetable oils are rich in Vit E; -and -tocopherols in wheat germ oil, - and -tocopherols in cotton seed oil and -tocopherol in soya bean oil. (II) Animals: Liver, egg yolk, milk and colostrum, contain small amount of Vit E. Function: 1. Non-specific anti-oxidant activity 2. Antisterity: Specially in rats Deficiency: The signs of human Vit E deficiency include: sensitivity of erythrocytes to peroxide and the appearance of abnormal cellular membrane which lead to anaemia (maturation arrest). Requirements: 30mg/day. - 52 - Biochemistry first year First term Minerals necessary in human nutrition are classified according to body needs into 2 groups 1- Major: Those that are required by the body in amounts of more than 100mg/dl. They include : calcium, phosphorus, magnesium, Sodium… etc. 2- Trace elements: which are required by the body in amounts less than 100 mg/dl. They include iron, iodine, zinc, fluoride…. etc. Calcium Calcium is the predominant mineral element in the body. The body of an adult male nearly contains 1-1.2 kg of calcium. Distribution of calcium in the body: About 99% of the body calcium is in the skeleton in which calcium is deposited in bone and has a structure essential to normal calcification. The major form of inorganic constituent of the bone is comprised of crystalline calcium phosphate resembling the mineral hydroxyapetit