Fundamentals Of Plant Biochemistry And Biotechnology PDF

Fundamental of Plant Biochemistry and Biotechnology Course Name : FUNDAMENTAL OF PLANT BIOCHEMISTRY AND BIOTECHNOLOGY 2  Fundamental of Plant Biochemistry and Biotechnology AME 111 - 11...

Fundamental of Plant Biochemistry and Biotechnology Course Name : FUNDAMENTAL OF PLANT BIOCHEMISTRY AND BIOTECHNOLOGY 2  Fundamental of Plant Biochemistry and Biotechnology AME 111 - 111 Fundamentals of Plant Biochemistry and Biotechnology A-Theory S.No Topics No. of Lec. 1 Importance of Biochemistry; Properties of Water, pH and 1 Buffer 2 Carbohydrate: Importance and classification; Structures of 3 Monosaccharides, Reducing and oxidizing properties of Monosaccharides, Mutarotation 3 Structure of Disaccharides and Polysaccharides 1 4 Lipid: Importance and classification; Structures and 2 properties of fatty acids 5 Proteins: Importance of proteins and classification; 2 Structures, titration and zwitterions nature of amino acids 6 Structural organization of proteins 1 7 Enzymes: General properties; Classification; Mechanism of 2 action 8 Michaelis & Menten and Line Weaver Burk equation & plots 1 9 Nucleic acids: Importance and classification 1 10 Structure of Nucleotides, A, B & Z DNA 2 11 RNA: Types and Secondary & Tertiary structure 2 12 Metabolism of carbohydrates: Glycolysis, TCA cycle, 3 Glyoxylate cycle, Electron transport chain 13 Metabolism of lipids: Beta oxidation, Biosynthesis of fatty 2 acids 14 Concepts and applications of plant biotechnology 1 15 Nutritional Requirement in Tissue Culture 1 15 Techniques in in vitro culture: Callus culture, Organ culture, 2 cell suspension culture, embryo culture, anther and pollen culture, ovule culture and their applications 16 Micro-propagation methods 1 17 Organogenesis and somatic embryogenesis 1 18 Synthetic seeds and their significance 1 19 Somatic hybridization and cybrids 1 20 Application in crop improvement of somatic hybrids and 1 cybrids 21 Somaclonal variation and its use in crop improvement; Cryo- 1 preservation 22 Introduction to recombinant DNA methods 1 23 Physical (Gene gun method), chemical (PEG mediated) and 2 Agrobacterium mediated gene transfer methods 24 Transgenics and its importance in crop improvement 2 25 PCR techniques and its applications 1 26 RFLP, RAPD, SSR 2 27 Marker Assisted Breeding in crop improvement 2 28 Biotechnology regulations 1 Total lectures of theory 44 3 Fundamental of Plant Biochemistry and Biotechnology AME 111 Introduction of Biochemistry Biochemistry comprises study of chemical nature of living organisms and their relationship with their environment. It tries to explain life processes at molecular level; the processes by which an exchange of chemical substances takes place between the living organisms and the environment the processes by which the absorbed materials are utilized for synthetic reactions leading to growth and replenishment of tissues and multiplication of the cell and the species the metabolic breakdown of the materials to supply energy for all the above and the mechanisms which regulate with precision all these processes. All these studies come under the purview of biochemistry. Correlation of biological functions and molecular structures is the central theme of biochemistry. Biochemistry includes various aspects of organic chemistry, physical chemistry, physics, biology and other basic disciplines. It is also interrelated with physiology, microbiology, medicine and agriculture. Importance of biochemistry During the early part of the twentieth century, the central theme of biochemistry was the development of the field of intermediary metabolism that is the elucidation of the pathways for the synthesis and degradation of the constituents of living organisms. Although studies concerned with intermediary metabolism continue to be important, at present, biochemical research may be classified into the following major areas: 1. Composition and characteristics of chemical compounds of living organisms. 2. Cell ultra structure. 3. Cellular control mechanisms. 4. Physical chemistry of bio macromolecules. 5. Structure-function, kinetics, regulation and mode of action of enzymes. 6. Intermediary metabolism. 7. Bioenergetics particularly the mechanisms of formation of adenosine triphosphate (ATP) in the process of oxidative phosphorylation. 8. The molecular basis for genetic and developmental phenomena. 9. The molecular basis for physiological phenomena including nerve conduction, muscle contraction, vision and transport across membrane 10. Role, transformation and requirement of nutrients in plants, animals and other organisms. 11. Chemistry of inheritance: structure-function and regulation of gene expression. 4 Fundamental of Plant Biochemistry and Biotechnology AME 111 Contribution of biochemistry in various field of Biology Several research done in field of biochemistry, microbiology, molecular genetics, cell biology and recombinant DNA has led to the development of 'biotechnology'. In 1981, the European Federation of Biochemistry defined this branch of science as "the integrated use of biochemistry, microbiology and chemical engineering in order to achieve the technological application of the capacities of microbes and culture cells". A recent off- shoot of biotechnology research is genetic engineering, which involves gene splicing and recombinant DNA cloning. Some of the applications of genetic engineering and biotechnology in various fields are listed below: 1. Agriculture Improvement of crop plants for higher photosynthetic efficiency, nutrient and water uptake, biological nitrogen fixation, nutritional quality of cereals, resistance to pests and diseases, plant cell and tissue culture and improvement of animal stock for desirable characteristics 2. Chemical industry Transformation of substances by biocatalysts to products such as biopolymers, antibiotics, alternative structural materials to plastics 3. Energy industry Production of new fuel sources and improved efficiency of energy recovery from existing ones. 4. Food industry Production of colourants, sweeteners, preservatives, etc 5. Fermentation Production of beer, wine, alcohols, amino acids, vitamins, industry etc., 6. Public health Production of vaccines, drugs, growth hormones, interferons monoclonal antibodies, etc., use of bacteria in waste water treatment and recycling and pollution control 5 Fundamental of Plant Biochemistry and Biotechnology AME 111 Properties of Water, pH and Buffer Water is the most abundant substance in living systems, making up 70% or more of the weight of most organisms. The attractive forces between water molecules and the slight tendency of water to ionize are of crucial importance to the structure and function of biomolecules. The water molecule and its ionization products, H+ and OH-, profoundly influence the structure, self-assembly and properties of all cellular components, including proteins, nucleic acids and lipids. The noncovalent interactions responsible for the strength and specificity of “recognition” among biomolecules are decisively influenced by the solvent properties of water, including its ability to form hydrogen bonds with itself and with solutes. Properties of water Water is a polar molecule. It has hydrogen bonding potential It has Specific heat, heat of vaporization Nucleophilic Ionization Water is an ideal biological solvent Water is a polar molecule It is a polar molecule: Atoms are held together by polar covalent bonds. This gives the oxygen as light negative charge (δ-) and each hydrogen as light positive charge (δ+). Hydrogen atoms are attached to the oxygen at an angle of 104.5o. Hydrogen bonding gives water its unusual properties Each water molecule can form up to four hydrogen bonds (with the oxygen accepting two hydrogen bonds, and with each hydrogen acting as an hydrogen bond donor). The arrangement is roughly tetrahedral. The H–O–H bond angle does not allow the formation of a perfect tetrahedron. (Note that in actual water, the average number of hydrogen bonds per water molecule is less than four, probably due to geometry constraints and entropic effects.) The ability of the water molecule to participate in extensive hydrogen bonding networks is responsible for most of the unusual bulk properties of water, including its high 6 Fundamental of Plant Biochemistry and Biotechnology AME 111 melting and boiling points, ∆H of vaporization, and surface tension. A look at the electron structure of the H2O molecule reveals the cause of these intermolecular attractions. Each hydrogen atom of a water molecule shares an electron pair with the central oxygen atom. The geometry of the molecule is dictated by the shapes of the outer electron orbitals of the oxygen atom, which are similar to the sp3 bonding orbitals of carbon. These orbitals describe a rou gh tetrahedron, with a hydrogen atom at each of two corners and unshared electron pairs at the other two corners. The H-OH bond angle is 104.5o, slightly less than the 109.5o of a perfect tetrahedron because of crowding by the nonbonding orbitals of the oxygen atom. The oxygen nucleus attracts electrons more strongly than does the hydrogen nucleus (a proton); that is, oxygen is more electronegative. The sharing of electrons between H and O is therefore unequal; the electrons are more often in the vicinity of the oxygen atom than of the hydrogen. The result of this unequal electron sharing is two electric dipoles in the water molecule, one along each of the H-O bonds; each hydrogen bears a partial positive charge and the oxygen atom bears a partial negative charge equal to the sum of the two partial positives. As a result, there is an electrostatic attraction between the oxygen atom of one water molecule and the hydrogen of another (Fig. 2–1c), called a hydrogen bond. Hydrogen bonds are relatively weak. Those in liquid water have a bond dissociation energy (the energy required to break a bond) of about 23 kJ/mol, compared with 470 kJ/mol for the covalent O-H bond in water or 348 kJ/mol for a covalent C-C bond. The hydrogen bond is about 10% covalent, due to overlaps in the bonding orbitals, and about 90% electrostatic. Water as solvent Hydrogen bonds between water molecules provide the cohesive forces that make water a liquid at room temperature and that favor the extreme ordering of molecules that is typical of crystalline water (ice). Polar biomolecules dissolve readily in water because they can replace water-water interactions with more energetically favorable water-solute interactions. In contrast, nonpolar biomolecules interfere with water-water interactions but 7 Fundamental of Plant Biochemistry and Biotechnology AME 111 are unable to form water-solute interactions consequently, nonpolar molecules are poorly soluble in water. In aqueous solutions, nonpolar molecules tend to cluster together. Hydrogen bonds and ionic, hydrophobic (Greek, “water-fearing”), and van der Waals interactions are individually weak, but collectively they have a very significant influence on the three-dimensional structures of proteins, nucleic acids, polysaccharides, and membrane lipids. Ionization of water Water spontaneously ionizes to release free protons and free hydroxyl ions. In aqueous media, free H+ doesn’t typically exist. Instead, the dissociated proton is shared between many other water molecules to form transient H3O+ and related species. H2O H+ + OH– For simplicity, in the following discussion the symbol H+ will be used to reflect the shared ion. Aqueous solutions do contain free hydroxyl ions, although these ions, as with all ions in aqueous solution, have a hydration shell K= [H+] [OH–] [H2O] However, in aqueous solution, the concentration of H2O is so high that it is not significantly altered by the extremely small amount of dissociation that occurs. The effectively constant water concentration is generally included in the equilibrium K term to simplify the calculations. Kw = [H+][OH–] = 10 -14M2 In pure water, therefore, [H+] = [OH–] = 10 -7M To simplify discussion another term was introduced to describe small concentrations of hydrogen ions: pH, which is defined as the negative log of the hydrogen ion concentration. Neutral is defined as pH 7.0, because this is the pH for water with no additional ions present. Because pH is a log function, a change of one pH unit (e.g., from pH 7 to pH 6) corresponds to a 10-fold change in hydrogen ion concentration. Weak acids Unlike strong acids, some compounds capable of releasing protons do not completely ionize in aqueous solution. For example, acetic acid also dissociates to yield a free proton and acetate ion (the conjugate base of acetic acid): 8 Fundamental of Plant Biochemistry and Biotechnology AME 111 Again, this process reaches equilibrium, with the dissociation constant given by: For acetic acid, Ka = 1.75 x 10 -5M; this indicates that only a small proportion of the acetic acid will dissociate. As with hydrogen ion concentrations, this dissociation constant value is somewhat clumsy to deal with, and therefore, as with pH, it is frequently worth using the negative log of the Ka: pKa = 4.75. By contrast, HCl has a pKa of -7. This illustrates a general principle: a higher pKa corresponds to a higher affinity for protons. To determine the pH, the equilibrium equation can be rearranged to solve for hydrogen ion concentration: Taking the negative log of both sides gives: This is the same as: which is the classic Henderson-Hasselbalch equation. For acetic acid (and for all weak acids), when [Ac–] = [HAc] the solution pH is equal to the pKa. The Henderson- Hasselbalch equation can be used to solve for the relative amounts of acid and conjugate base present at any pH. 9 Fundamental of Plant Biochemistry and Biotechnology AME 111 Buffers Solutions containing mixtures of weak acids and their conjugate bases resist changes in pH due to the addition of either hydrogen ions or hydroxide ions. These solutions are buffers. Buffers are critical for maintenance of pH control both in experiments in vitro and in physiological systems (although physiological systems also use active mechanisms to control pH). Weak acids have the ability to act as buffers. Because the conjugate base of a weak acid has a relatively high affinity for hydrogen ions, it will absorb any hydrogen ions added to a solution; alternatively, if the pH increases, the weak acid can release hydrogen ions. Adding HCl to water causes a rapid drop in pH to a final pH of 1. On the other hand, in the presence of Tris base (a commonly used buffer compound), the decrease in pH is markedly attenuated, with the final pH being 2 pH units higher. In the titration shown below for 0.1 M Tris, the solution pH changes from a high value to a low value upon addition of the strong acid HCl. The inflection point at 0.5 equivalents of strong acid corresponds to the pKa of the protonated form of Tris. As is apparent in the plot, between about 0.05 and 0.95 equivalents (corresponding to ±1 pH around the pKa), the system resists change in pH. (Note that, to a very good approximation, all of the added protons can be assumed to become bound by the conjugate base; the pH change then results from the altered ratio of conjugate base to weak acid.) In principle, the Henderson-Hasselbalch equation can be used to calculate the pH of a buffer given the ratio of weak acid and conjugate base, or to calculate the amount of weak acid and conjugate base that must be added to a solution to obtain a desired pH. Because, in most cases, real solutions deviate from ideal behavior, most biochemists obtain the desired pH by titrating a solution while directly measuring pH using a pH meter. The Henderson-Hasselbalch allows an estimate of the amount of strong acid or strong base that must be added to obtain the correct pH, which tends to make buffer preparation less tedious. 10 Fundamental of Plant Biochemistry and Biotechnology AME 111 Carbohydrates Based on chemical constitution, the carbohydrates or saccharides are most simply defined as polyhydroxy aldehydes or ketones and their derivatives. But this definition is not entirely satisfactory. Because, presence of free carbonyl group (>C=O) in the simple carbohydrate molecule is not true. Because carbonyl compound reacts with an alcohol to form a hemiacetal. In carbohydrate,we have an aldehyde group which combine with an alcoholic –OH of the same molecule to form an interal hemiacetal and by elimination of H2O between the hemiacetal –OH group of two sugar molecules are formed. So that the definition of carbohydrate may be improved by a polyhydroxy compound that has an aldehyde or a ketone function present, either free or as hemiacetal or acetal. Carbohydrates are composed of carbon, hydrogen and oxygen possessing a common empirical formula Cn(H2O)n. Occurrence Carbohydrates are the most abundant of all biochemical compounds and constitute more than 50 percent of the total biochemical matter. They are widely distributed in plants, animals and microbes. They are synthesized in green plants and algae from water and CO2 using solar energy in a process called photosynthesis. Physiological role and biological importance The carbohydrates serve many functions in the living organisms. Some of their vital functions are: 1. Chief source of energy (4 kcal/g). 2. Reserve or storage forms of energy in plants (starch, inulin) and animals (glycogen). 3. Structural elements in plant cell wall (cellulose), exoskeleton of some insects and crustacea (chitin), cell walls of certain microorganisms (peptidoglycans) and skin and connective tissues of animals (mucopolysaccharides). 4. Important components of nucleic acids, co-enzymes and flavoproteins (for example, ribose). 5. They are involved in cell recognition, contact inhibition and also have antigenic 11 Fundamental of Plant Biochemistry and Biotechnology AME 111 properties of blood group substances. Classification The carbohydrates can be classified into three main groups as: a) monosaccharides, b) oligosaccharides and c) polysaccharides, based on number of monomeric sugar units present. Monosaccharides are the simplest sugars consisting of single polyhydroxy aldehyde or ketone group that cannot be hydrolyzed into smaller units under reasonable mild conditions. They serve as the building-blocks for the more complex sugars. Oligosaccharides (Greek Oligo 'few') contain from two to ten monosaccharide units joined through glycosidic linkage or bond. They are hydrolysable into constituent monosaccharide units. Polysaccharides are polymers of monosaccharide units joined in long linear or branched chains through glycosidic bonds. Hydrolysis of polysaccharides yields many units of constituent monosaccharides. Polysaccharides have two major biological functions: a) as a storage form of fuels and b) as structural elements in living organisms. 1. Monosaccharides (simple sugars) Monosaccharides, also called as simple sugars have the empirical formula (CH2O)n, where n=3 or larger number. They contain a short chain of carbon atoms with one carbonyl group, each of the remaining carbon atoms bearing a hydroxyl group. If the carbonyl group is an aldehyde (-CHO) the sugar is called as an aldose (name ends in 'ose') and if a ketone (C=O) it is a ketose (usually ends in 'ulose'). The simplest monosaccharides are the 3-carbon trioses glyceraldehyde and dihydroxyacetone. Glyceraldehyde is an aldotriose; dihydroxyacetone is a ketotriose. Other monosaccharides are tetroses (four carbons), pentoses (five carbons), hexoses (six carbons), heptoses (seven carbons) and octoses (eight carbons). Each exists in two series, 12 Fundamental of Plant Biochemistry and Biotechnology AME 111 ie., aldotetroses and ketotetroses; aldopentoses and ketopentoses; aldohexoses and ketohexoses, etc. Hexoses (both aldoses and ketoses) are the most abundant among monosaccharides. Glucose (aldohexose) is the most abundant monosaccharide; serves as the major fuel for most organisms and the- basic building-block of the many oligo- and polysaccharides. However, aldopentoses are important components of nucleic acids (for example, ribose) and various polysaccharides (for example, xylose and arabinose). Trioses, tetroses and heptoses are important intermediates in carbohydrate metabolism. Table: Some important monosaccharides. Name Major source Function Type No. of carbons Xylose Hydrol,ysis of wood, Constituent of Aldopentose 5 (wood straw, seed hulls straw, seed hulls sugar) wood, Arabinose Hydrolysis of gum Constituent of Aldopentose 5 arabic, cherry tree gum arabic gum, pectin Ribose Hydrolysis of nucleic Constituent of Aldopentose 5 acids nucleic acids Glucose Ripe fruits, sweet Energy source Aldohexose 6 (dextrose) corn, honey, blood, egg yolk Mannose Hydrolysis of Constituent of Aldohexose 6 mannans mannans Galactose Hydrolysis of lactose Constituent of Aldohexose 6 milk sugar Fructose Honey, sweet fruits Energy source Ketohexose 6 (levulose) General properties Some of the general properties of monosaccharides are summarized below: b) Monosaccharides are polyhydroxy aldehydes or ketones and their derivatives having either a potentially free aldehyde or a ketone group. c) Simplest form of carbohydrates which cannot be hydrolyzed to other sugar units under reasonably mild chemical conditions. d) Generally monosaccharides are white crystalline solids, insoluble in ether, sparingly soluble in alcohol but readily soluble in water. e) Most of them have a sweet taste and char when heated. f) Those with potentially a free aldehydic or a ketonic group are able to reduce metal ions under alkaline conditions. Hence, they are excellent reducing agents. 13 Fundamental of Plant Biochemistry and Biotechnology AME 111 g) Amphoteric nature i.e., they are capable of reacting as weak acids or weak bases with strong acids or alkalies to form salts. Stereochemistry All the monosaccharides except dihydroxyacetone contain one or more asymmetric carbon atom(s) i.e., a single carbon atom having four different substituents and thus are chiral molecules. Subcompounds are capable of existing in two or more isomeric forms that are non-superimposable mirror images of each other. Such com· pounds exist in right- handed forms and are called chiral (hand) compounds. This phenomenon is called chirality (handedness). Glyceraldehyde contains only one asymmetric carbon atom (carbon atom, 2) and therefore can exist as two different stereoisomers, i.e., as D- and L- glyceraldehyde. The symbols D- and L- designate the absolute configuration of an isomer and not the sign of rotation of plane-polarized light. The structure with -OH group on the right and –CHO group on the top of the asymmetric carbon atom is designated as D-glyceraldehyde. The structure in which the -OH group is to the left and -CHO group on the top is designated as L-glyceraldehyde. The D- and L-glyceraldehydes are used as reference or parent compounds for designating the absolute configuration of all stereoisomeric compounds. The term configuration refers to the special arrangement of the atoms in a molecule resulting from the double bonds and/or chiral centers. Configurational isomers cannot be interconverted without breaking one or- more covalent bonds. Enantiomers Aldoses and ketoses of the L-series are mirror-images of their D-counterparts as shown in Fig. 2.5. These two D- and L- forms of a sugar are known as enantiomers. L- sugars are found in nature, but they are not so abundant as D-sugars. Diastereoisomers Two sugars having the same molecular formulae but not the mirror images of each other are known as diastereoisomers. e.g. D-glucose and D-mannose. All these sugars are not mirror images of each other. Epimers Two sugars differing only in the configuration around one specific carbon atom are called epimers of each other. Thus, D-glucose and D-mannose are epimers with respect to carbon atom 2, and D-glucose and D-galactose are epimers with respect to carbon atom 4. Optical activity 14 Fundamental of Plant Biochemistry and Biotechnology AME 111 All the monosaccharides except dihydroxyacetone contain one are more asymmetric carbon atom(s) and thus are optically active. Optical activity refers to the ability of a compound in solution to rotate the plane of polarization of plane-polarized light when observed in a polarimeter. Optical activity is shown by all compounds capable of existing in two forms that are non-superimposable mirror images of each other. The optical activity is expressed quantitatively as the specific rotation: Where, α = specific rotation in degrees at temperature usually 25 oC and the wavelength of the light employed (usually the D line of sodium) is 589.3nm. If the rotation of the beam of plane-polarized light is clockwise (to the right or rectus as the observer looks towards the light source), the enantiomer is designated as dextrarotatory (dextro, 'd' or '+' symbols) and if it is anticlockwise (to the left or sinister), the enantiomer is designated as levorotatory (levo, '1' or '-' symbols). For example, the specific rotation of a-D-glucose is + 112.2o (dextrarotatory) and that of D- fructose is -930 (levorotatory). Thus, the symbol '+' and '-' refer to the direction of rotation of the beam of plane-polarized light but not the absolute configuration. The D-and L-stereoisomers of any given compound have identical physical properties and identical chemical reactivities, with two exceptions: (a) they rotate the plane of plane- polarized light equally but in opposite directions and (b) they react at different rates with reagents that are themselves asymmetric. The equimolar mixture of the D- and L- stereoisomers, known as racemic mixture or racemate (designated as D L-) is optically inactive as the asymmetric carbon atom passes through a symmetrical intermediate during chemical reaction. Structural aspects Ring structure and mutarotation In aqueous solution, many monosaccharides act as if they have one more asymmetric center than is given by the open chain structural formulae. D-glucose may exist in two different isomeric forms differing in specific rotation, α-D-glucose (Crystallizes from a concentrated aqueous solution at 30°C , melting point 146°C), for which [α]D20 = + 112°, and β-D-glucose (Crystallizes from hot glacial acetic acid solution, melting point 148- 150°C), for which [α]D20 = +19°. These two sugars do not differ in elementary composition but differ in physical and chemical properties. When the α- and β-isomers of D-glucose are dissolved in water, the optical rotation of each gradually changes with time 15 Fundamental of Plant Biochemistry and Biotechnology AME 111 and approaches a final equilibrium value of [α]D20 = +53°. This change, called mutarotation is due to the formation of an equilibrium mixture consisting of about one- third α-D-glucose and two thirds β-D-glucose at 20°C. Although carbohydrates are formally aldehydes or ketones, a sugar like glucose does not readily answer the normal reactions of aldehydes as would be expected; this is because of their existence as cyclic hemiacetals or hemiketals. Aldehydes can react with an alcohol to form a hemiacetal; similarly ketones can react with an alcohol to form a hemiketal. Anomers From various chemical considerations it has been deduced that the α- and β-isomers of D-glucose are not open-chain structures in aqueous solution but six-membered ring structures formed by the reaction of the alcoholic hydroxyl group at carbon atom 5 with the aldehydic carbon atom 1 to form a hemiacetal which renders an other chiral center at carbon atom 1, also known as carbonyl carbon atom or anomeric carbon atom. Isomeric forms of monosaccharides that differ from each other only in configuration about the carbonyl carbon atom are known as anomers. Thus, D-glucose will have two anomers designated as α-D-glucose and β-D-glucose. As hemiacetal or hemiketal formation is reversible, if one of the anomers is dissolved in water, an equilibrium mixture of the two anomers results. This interconversion between the two anomers is due to mutarotation. The cyclic hemiacetal formation in the case of glucose by the reaction of the alcoholic hydroxyl group at carbon 5 with the aldehydic carbon atom 1 results in formation of six-membered ring. The six-membered ring forms of sugars are called pyranoses because they are derivatives of the heterocyclic compound pyran. Thus, the systematic name for the ring form of α-D- glucose is α-D-glucopyranose. In the case of fructose, the hemiketal is formed by reaction of the hydroxyl group on carbon atom 5 with the carbonyl group at carbon atom 2 to yield a five-membered ring. The five-membered ring forms of sugars are called furanoses as they are derivatives of heterocyclic compound, furan (Fig. 2.8) as suggested by Haworth. The systematic name 16 Fundamental of Plant Biochemistry and Biotechnology AME 111 for the ring form of α-D-fructose is α-D-fructofuranose. 2. Oligosaccharides These sugars consist of a short chain of 2 to 10 monosaccharide units linked by the glycosidic bond (s) with the elimination of water molecule (s). The glycosidic bond is formed most frequently between the anomeric carbon of one sugar residue and a hydroxyl group of the other sugar residue. Depending on the number of monosaccharide units that are linked, the oligosaccharides are further classified as disaccharides (two sugar units), trisaccharides (three sugar units), tetrasaccharides (four sugar units), etc. Amongst these, disaccharides are the most important class because of their biological role and relative abundance in natural products. 2.1.Disaccharides (C12H22O11). These are a group of compound sugars composed of two monosaccharides linked by the glycosidic bond with the elimination of one molecule of water. General properties 1. Those with potentially a free aldehyde or a ketone group can reduce Fehling's solution, hence are called reducing disaccharides. 2. The reducing disaccharides have most of the properties of monosaccharides i.e., they can form osazones and show mutarotation, etc. 3. Disaccharides can be hydrolyzed into their constituent monosaccharide units unlike monosaccharides. 4. Some disaccharides may exist in white crystalline solids and are soluble in water and sweet in taste. 5. Disaccharides are not fermented by yeast directly but they are first hydrolyzed to constituent monosaccharides which in turn are fermented. The most abundant disaccharides in nature are maltose, sucrose and lactose. 2.1.1 Maltose It is a disaccharide formed by linking two units of α-D-glucose through α-1,4 glycosidic bond with the elimination of one molecule of water '(Fig. 2.20). It is a reducing sugar since the -OH group bound to carbon 1 of the glucose residue 17 Fundamental of Plant Biochemistry and Biotechnology AME 111 is free and can exist in the aldehyde form. It exhibits mutarotation since it exists in both α- and β-forms. Malt prepared from sprouting barely, is an excellent, source of maltose. 2.1.2 Sucrose (α-D-glucopyranosyl-β-D-fructofuranoside) Sucrose or cane sugar or beet sugar or saccharose or invert sugar is a disaccharide made up of one molecule each of α-D- glucose and β-D-fructose, the linkage involving the potential aldehyde group of carbon atom 1 of glucose and the ketonic group of the carbon atom 2 of fructose (β,2- >1) linkage. It is a non-reducing.sugar because of the absence of a potentially free aldehyde or ketonic group and forms no osazone. As it does not exist in α- and β-forms, it fails to exhibit mutarotation. It is hydrolyzed by acid or enzyme sucrase (invertase) into glucose and fructose. The specific rotation of sucrose is +66.5° and after hydrolysis, the specific rotation of the mixture is -19.84°. Such a change in specific rotation. from dextro- to levorotatory nature is called 'inversion' and hence the name 'invert sugar'. The reason for the inversion is that fructose is more strongly levorotatory (-93° than glucose which is dextrarotatory (+52.5°). It is the most abundant oligosaccharide and is ubiquitous in plants. It is generally manufactured from sugarcane and sugar beet. 2.1.3 Lactose (4-O-β-D-galactopyranosyl-D-glucopyranose) Lactose or milk sugar is made up of β-D- galactose and α-(in α-form) or β-(in β-form) D-glucose through β-1, 4 glycosidic bond (Fig. 2.20). It is a reducing sugar, exhibits mutarotation and forms osazone. It reduces Fehling's solution but not Barfoed's reagent and thus can be distinguished from other reducing disaccharides. It is hydrolyzed by the enzyme lactase into its constituent hexoses. It does not ferment as easily as glucose and hence makes an ideal constituent of milk of mammals (about 5 g/100 ml milk). It is not produced in plants. 2.1.4 Cellobiose (4-O-β-D-glucopyranosyl-D-glucopyranose) It is a partial hydrolytic product of cellulose, made up of two glucose units joined through β-1, 4 linkage (Fig. 2.20). It is a reducing sugar. It is probably present in only trace 18 Fundamental of Plant Biochemistry and Biotechnology AME 111 amounts in nature and formed during the digestion of cellulose by the cellulases of microorganisms. 2.1.5 Trehalose (1-O-α-D-glucopyranosyl-1-α-D-glucopyranoside) It is made up of two glucose units linked through two anomeric carbon atoms. It is a non- reducing sugar. It is the major carbohydrate present in insects and fungi where it serves as a storage carbohydrate from which glucose may be obtained as required. 2.2 Trisaccharides (C18H34O17) A naturally occurring trisaccharide is raffinose [α-D-galactopyranosyl-O-( 1,6-α-D- glucopyranosyl-O-(1,2) –β-D-fructofuranoside] found in sugar beet, coffee and other plant materials. It is a non-reducing sugar. Melezitose[O-α-ODglucopyranosyl(1->3)-O-β-D-fructofuranosyl(2,1)-α-D glucopyranoside] is found in the sap of some coniferous trees. 2.3 Tetrasaccharides The important one among tetrasaccharides is the stachyose derived from raffinose. Stachyose consists of galactose-galactose-glucose-fructose monosaccharide sugars linked through α-1, 6, α-l, 6 and α-1, 2 glycosidic bonds, respectively. It occurs during germination of seeds. 3. Polysaccharides or glycans (C5H10O5)n These are complex carbohydrates which are polymerized anhydrides of a large but undetermined number of the simple sugars which are joined by glycosidic bonds. Those found in nature contain either five or six carbon monosaccharide units. The bulk of carbon found in nature exists in the form of polysaccharides. These are involved in the majority of biological processes although free monosaccharides and disaccharides occur in many biological fluids and plants. General properties Some of the important properties of polysaccharides are as follows: 1. Complex sugars of high molecular weight; polymers of several units of monosaccharides or either derivatives with linear or branched chains. 2. Upon hydrolysis by acids or enzymes, they are broken down into various intermediate products and finally into their consituent monosaccharides or their derivatives. 3. They are tasteless, apparently amorphous, some are crystalline 4. Mostly insoluble in cold water but form a sticky or gelatinous solutions 5. They differ in the nature of their recurring monosaccharides units, in the length of their chains and in the degree of branching 19 Fundamental of Plant Biochemistry and Biotechnology AME 111 Biological role Polysaccharides serve two main functions in the living organisms as: 1. Storage form of cellular fuel and 2. Structural elements in animal, plant and microbial systems Classification Polysaccharides can be classified in many ways A. Based on function 1. Structural polysaccharides: These polysaccharides serve as structural components of living organisms. e.g. cellulose (plant cell wall), chitin (exoskeleton of some insects), etc. 2. Storage/reserve/nutrient polysaccharides: These polysaccharides function as reserve or storage form of fuel in living organisms e.g. starch (plants), glycogen (animal cells) etc. B. Based on composition: 1. Homopolysaccharides: These are made up of single kind of monosaccharide residues or their derivatives. e.g. Starch, glycogen, cellulose, chitin, inulin, etc. 2. Heteropolysaccharides: These are made up of two or more different kinds of monosaccharide units or their derivatives. e.g. Hyaluronic acid, heparin, pectins, gums, mucilages, chondroitins, etc. Polysaccharides are often called as glycans. Those containing glucose are called as glycans (starch and glycogen); those containing mannose are called mannans and those containing galactose units are called galactans. Structural polysaccharides Cellulose It is the most abundant organic compound of our planet accounting for about 50 per cent of all carbon. It is the principal constituent of cell walls in higher plants forming the main structural element. It is a linear homopolymer of glucose units linked by β-1,4 glycosidic bonds. It is insoluble in water and all organic solvents. It dissolves in conc. H2SO4, on diluting the solution and boiling, glucose is formed as final product. Partial hydrolysis of cellulose yields cellobiose, a disaccharide. Cellulase, a β-glucosidase produced by many bacteria and fungi, hydrolyzes cellulose. The large amount of glucose present in cellulose is not available as a source of energy for humans due to the lack of enzymes capable of cleaving the β-1,4 bonds. However, ruminants can effectively use cellulose as they contain a large bacterial population in their rumen capable of hydrolyzing it. 20 Fundamental of Plant Biochemistry and Biotechnology AME 111 In plant cell walls, cellulose microfibrils are cemented together by other substances important among them being pectin and hemicellulsoe. Pectins contain arabinose, galactose and glacturonic acid while hemicelluloses are homopolymers of D-xylose linked by β- 1,4 bonds. The important sources of cellulose are cotton fibers (98%), jute (50-70%), wood (40-50%), algae and bacteria. Cellulose and its derivatives are widely used in textiles, films and plastics. Chitin It is a structural homopolysaccharide made up of N-acetyl glucosamine residues in β- 1,4 linkage. It is the principal structural polysaccharide present in the exoskeleton of crustaceous insects, earthworms and mollusks. It is the second most abundant organic substance on earth. 21 Fundamental of Plant Biochemistry and Biotechnology AME 111 Table: Structure and functions of some polysaccharides No. of Polymer Type Repeating unit monosaccharide Role unit Starch A linear polymer of D-glucose Amylose Homo 50-50000 residues in (α1→4) linkage. Glucose residues in amylopectin Energy storage: in plants chains are (α1→4); the branch Amylopectin Homo Up to 106 points (Occurring every 24 to 30 residues) are (α1→6) linkages. polymer of (α 1→4)-linked Glycogen Homo subunits of glucose, with (α 1→6)- Up to 50000 Energy storage: in bacteria and animal cells linked branches, D-glucose residues are in Structural: in plants, gives rigidity and Cellulose Homo Up to 15000 (β1→4)linkage strength to cell walls A homopolymer of N-acetyl-D- Structural: in insects, spiders, crustaceans, Chitin Homo glucosamine units in (β1→4) Very large gives rigidity and strength to exoskeletons linkage. (α1→6)-linked poly-D-glucose Structural: in bacteria, extracellular Dextran Homo Wide range with (α1→3) branches adhesive Hetero-; (β1→4)-linked N- Structural: in bacteria, gives rigidity and Peptidoglycane peptides acetylglucosamine and N- Very large strength to cell envelope attached acetylmuramic acid residues D-galactose (β1→4)-linked to 3,6- anhydro-L-galactose that are Agarose Hetero- 1000 Structural: in algae, cell wall material joined by (α1→3) glycosidic links to form polymer Hyaluronate Structural: in vertebrates, extracellular Hetero-; D-glucuronic acid(β1→3) and N- (a glycosamine Up to 100000 matrix of skin and connective tissue; acidic acetylglucosamine (β1) -glycan) viscosity and lubrication in joints 22 Fundamental of Plant Biochemistry and Biotechnology AME 111. Peptidoglycan (murein) It is a structural heteropolysaccharide present in bacterial cell walls. The repeating unit of peptidoglycan is the muropeptide which is a disaccharide composed of N-acetyl-D- glucosamine (NAG) and N-acetyl muramic acid (NAMA) joined by a, β-1,4 glycosidic bond. NAMA consists of a NAG unit which has its C-3 hydroxyl group joined to the hydroxyl group of lactic acid by an ether linkage.. In the peptidoglycan the carboxyl group of each lactic acid moiety is in turn linked to a tetrapeptide consisting of L-alanine, D-isoglutamine, L-lysine and D-alanine (Fig. 2.21). The terminal D-alanine residue of the side chain of one polysaccharide chain is joined covalently with the peptide side chain of an adjacent polysaccharide chain, either directly as in E.coli or through a short conneting peptide, e.g. The pentaglycine in Staphylococcus aureus. The peptidoglycan structure of the bacterial cell wall is resistant to the action of peptide- hydrolyzing enzymes, which do not attack peptides containing D-amino acids. However, the enzyme lysozyme, found in tears and in egg white, hydrolyzes the β(1 4) glycosidic bonds of the polysaccharide backbone of the peptidoglycan structure. Reserve or storage polysaccharides Starch It is a principal storage homopolysaccharide of the plant kingdom, made up of D-glucose as repeating units. It is a mixture of two components amylose (about 20%) and amylopectin (about 80%) Amylose consists of long unbranched chains of D-glucose units which are linked by α-1, 4 glycosidic bonds. Its molecular wieght ranges from a few thousands of about 500,000. It gives blue colour with iodine due to the iodine-amylose complex in which iodine molecule is occupying a position in the interior of the helical coil. Amylopectin also has a backbone of α-1, 4 linked glucose units but in addition, branched through α-1, 6 linkages. The average length of branching is from 24 to 30 glucose residues. It gives a purple colour with iodine. Its molecular weight may range from 50,000 to 1,000,000. Both amylose and amylopectin can be hydrolyzed by the enzymes α- and β-amylases. α- amylases cleave α-1,4 linkages at random to give the mixture of maltose and glucose units while the β-amylases, present in plants remove maltose units succcesively from the non reducing end. The intermediate product left after the cleavage of starch by α and β amylases is 23 Fundamental of Plant Biochemistry and Biotechnology AME 111 called limit dextrins. Neither of these enzymes can hydrolyze α-1,6 linkages. Microbial glucomylase can act on both α-1, 4 and α-1, 6 likages of starch to yield glucose. Starch forms the major source of carbohydrates in the human diet and is of great economic importance. The important source of starch are seeds, fruits, tubers, bulbs and cereal grains varying from a few per cent of over 75 per cent. It is also found in some protozoa, bacteria and algae. Glycogen It is the storage homopolysaccharide in animals and is often called ‘animal starch’. It is present mainly in liver, skeletal muscle and in smaller amounts in all other tissues. It is stored in liver and muscles of animals and split to glucose in the liver to maintain proper concentration of glucose in the blood to furnish energy. The amount of glycogen present in the animal varies widely among the different tissues with diet and physiological state of the body. It is also aabundant in the mollusks while glycogens like polysaccharides are found in some bacteria. Glycogen is a branched chain of D-glucose units resembling amylopectin of starch. However, the branching through α-1, 6 linkages is more extensive than amylopectin, with 8- 10 glucose units between branching points. A glycogen molecule may contain as many as 30,000 glucose units. It is readily dispersed in water to form an opalescent solution which gives a reddish brown colour with iodine. It does not reduce Fehling’s solution. Insulin It is a storage polysaccharide in the Compositae family (artichokes, dahlias, dandelions, etc). It is a homopolymer made of D-Fructose units linked by β(2→1) bonds. Dextrans These are storage polysaccharides of some yeasts and bacteria. They consist of D-glucose units joined by α-1, 6 glycosidic bonds primarily with cross linkages through α-1,2 and and α- 1, 3 linkages. Glycoproteins Glycoproteins are molecules composed of covalently joined protein and carbohydrates. The carbohydrate is attached to the polypeptide chains of the protein in a series of reactions that are enzymatically catalyzed after the protein component is synthesized. Glycoproteins in cell membranes apparently have an important role in the group behavior of 24 Fundamental of Plant Biochemistry and Biotechnology AME 111 cells and other biological functions of the membrane. They form a major part of the mucus that is secreted by epithelial cells, where they perform an important role in lubrication and in the protection of tissues lining the body's ducts. Many other proteins secreted from cells into extracellular fluids are glycoproteins. These proteins include hormone proteins found in blood, such as follicle stimulating hormone (FSH), lutenizing hormone (LH), chorianic gonadotropin; and plasma proteins such as immunoglobulins etc. Glycoproteins are also one of the major components of the cell coats of higher organisms. The carbohydrate percent within glycoproteins is highly variable. Some glycoproteins such as IgG contain low amounts of carbohydrate (4 %). Human ovarian cyst glycoprotein is composed of 70 per cent carbohydrate and human gastricglycoprotein is 82 per cent carbohydrate. Glycoproteins having a very high content of carbohydrate are called proteoglycans. ***** 25 Fundamental of Plant Biochemistry and Biotechnology AME 111 Amino acid and Protein All proteins are formed from 20 different amino acids. All the amino acids have trivial or common names based on the source from which they were first isolated or based on their properties. e.g. Asparagine was named so, as it was isolated from asparagus and glycine was so named because of its sweet taste (Greek:'glykos' meaning sweet). All the 20 amino acids, except proline, found in proteins have an amino group and a carboxyl group attached to the same carbon atom, namely the α-carbon. They differ only in the side chains (R groups). The 20 amino acids found in proteins are referred as the standard or normal or protein amino acids. There are many other amino acids found in nature but do not occur in proteins. They are referred as non-protein amino acids. Classification of protein amino acids The protein amino acids are classified according to the chemical nature of their R groups as aliphatic, aromatic, heterocyclic and sulphur containing amino acids. More meaningful classification of amino acids is based on the polarity of the R groups. The polarity of the R groups varies widely from totally non-polar to highly polar. The 20 amino acids are classified into four main classes whose structures, three-letter and one-letter symbols are given below a) Amino acids with non-polar or hydrophobic, aliphatic R groups This group of amino acids includes glycine, alanine, valine, leucine, isoleucine and proline. The hydrocarbon R groups are non-polar and hydrophobic. The side chains of alanine, valine, leucine and isoleucine are important in promoting hydrophobic interactions within protein structures. The minimal steric hindrance of the glycine side chain (hydrogen) allows more flexibility than other amino acids. On the other hand, the imino group of proline is held in a rigid conformation and reduces the structural flexibility of the protein. b) Amino acids with non-polar aromatic R groups This group includes phenylalanine, tyrosine and tryptophan. All these amino acids participate in hydrophobic interactions, which is stronger than aliphatic R groups because of stacking one another. Tyrosine and tryptophan are more polar than phenylalanine due to the presence of hydroxyl group in tyrosine and nitrogen in the indole ring of tryptophan. The absorption of ultraviolet (UV) light at 280 nm by tyrosine, tryptophan and to a lesser extent by 26 Fundamental of Plant Biochemistry and Biotechnology AME 111 phenylalanine is responsible for the characteristic strong absorbance of light by proteins. This property is exploited in the characterization and quantification of proteins. b) Amino acids with polar, uncharged R groups This group of amino acids includes serine, threonine, cysteine, methionine, asparagine and glutamine. The hydroxyl group of serine and threonine, the sulphur atom of cysteine and methionine and the amide group of asparagine and glutamine, contribute to the polarity. The R groups of these amino acids are more hydrophilic than the non-polar amino acids. c) Amino acids with charged R groups Acidic: The two amino acids with acidic R groups are aspartic and glutamic acids. These amino acids have a net negative charge at pH 7.0. Basic: This group includes lysine, arginine and histidine. The R groups have a net positive charge at pH 7.0. The lysine has a second -amino group; arginine has a positively charged guanidino group; and histidine has an imidazole group. Physical Properties of amino acids Amino acids are white crystalline substances. Most of them are soluble in water and insoluble in non-polar organic solvents (e.g., chloroform and ether). Aliphatic and aromatic amino acids particularly those having several carbon atoms have limited solubility in water but readily soluble in polar organic solvents. They have high melting points varying from 200-300oC or even more. They are tasteless, sweet or bitter. Some are having good flavour. Sodium glutamate is a valuable flavouring agent and is used in the preparation of certain dishes and sauces. Amphoteric nature of amino acids Ø Amino acids are amphoteric compounds, as they contain both acidic (COOH) and basic (NH2) groups. Ø They can react with both alkalis and acids to form salts. Ø In acid solution amino acids carry positive charges and hence they move towards cathode in an electric field. Ø In alkaline solution, the amino acids carry negative charges and therefore move towards anode. Ø When an amino acid is dissolved in water, it exists as inner salt carrying both positive and negative charges.This occurs as a result of dissociation of carboxyl group to release the 27 Fundamental of Plant Biochemistry and Biotechnology AME 111 H+ ion, which passes from the carboxyl to the amino group. The amino acids possessing both positive and negative charges are called Zwitterions. The zwitterion reacts as an acid with a base by liberating a proton (H+) from the NH3+ group and as a result possesses a net negative charge. On the other hand, zwitterions reacts with an acid as base, combining with the proton (H+) of the acid resulting in the formation of a compound having a net positive charge. These reactions are reversible. The pH at which the amino acid has no tendency to move either towards positive or negative electrode is called isoelectric pH or isoelectric point. At isoelectric pH, the amino acid molecule bears a net charge of zero. Isomerism Ø All amino acids except proline, found in protein are -amino acids because NH2 group is attached to the -carbon atom, which is next to the COOH group. Ø Examination of the structure of amino acids reveals that except glycine, all other amino acids possess asymmetric carbon atom at the alpha position. Ø Because of the presence of asymmetric carbon atom, amino acids exist in optically active forms. Ø For example, in the steric configuration for serine, the carboxyl group is written on the top, while the amino group is written to the left in the case of L-serine and to the right in the case of D-serine This distinction will hold good for all the amino acids having asymmetric carbon atoms. Ø 'D' and 'L' do not refer to the optical rotation, but to the steric configuration of amino group to the right and left side of the carboxyl group. 28 Fundamental of Plant Biochemistry and Biotechnology AME 111 Ø The direction of optical rotation of amino acid is indicated by the symbol + or -, which follows the designation 'D' or 'L'. Ø The steric configuration and optical rotation of an amino acid may be simultaneously expressed as D (+) or D (-) and L (+) or L (-). Ø L-forms are more common than D-forms and most of the naturally occurring amino acids are L-amino acids. Chemical properties a) Reactions due to amino group Reaction with formaldehyde (Formal titration) Ø Amino acid exists as zwitterion in aqueous medium. If an amino acid solution is treated with excess of neutralized formaldehyde solution, the amino group combines with formaldehyde forming dimethylol amino acid which is an amino acid formaldehyde complex. Ø Hence the amino group is protected and the proton released is titrated against alkali. Ø This method is used to find out the amount of total free amino acids in plant samples. Reaction with nitrous acid Nitrous acid reacts with the amino group of amino acids to form the corresponding hydroxyacids and liberate nitrogen gas. Reaction with ninhydrin Ninhydrin is a strong oxidizing agent. When a solution of amino acid is boiled with ninhydrin, the amino acid is oxidatively deaminated to produce ammonia and a ketoacid. The keto acid is decarboxylated to produce an aldehyde with one carbon atom less than the parent amino acid. The net reaction is that ninhydrin oxidatively deaminates and decarboxylates - amino acids to CO2, NH3 and an aldehyde. The reduced ninhydrin then reacts with the liberated ammonia and another molecule of intact ninhydrin to produce a purple coloured compound known as Ruhemann's purple. This ninhydrin reaction is employed in the quantitative determination of amino acids. Proteins and peptides that have free amino group(s) (in the side chain) will also react and give colour with ninhydrin. 29 Fundamental of Plant Biochemistry and Biotechnology AME 111 Reactions due to carboxyl group Decarboxylation Ø The carboxyl group of amino acids is decarboxylated to yield the corresponding amines. Thus, the vasoconstrictor agent, histamine is produced from histidine. Ø Histamine stimulates the flow of gastric juice into the stomach and the dilation and constriction of specific blood vessels. Ø Excess reaction to histamine causes the symptoms of asthma and various allergic reactions. Essential amino acids Ø Most of the prokaryotic and many eukaryotic organisms (plants) are capable of synthesizing all the amino acids present in the protein. But higher animals including man possess this ability only for certain amino acids. Ø The amino acids, which are needed for normal functioning of the body but cannot be synthesized from metabolic intermediates, are called essential amino acids. Ø These must be obtained from the diet and a deficiency in any one of the amino acids prevents growth and may even cause death. Ø Methionine, Arginine, Threonine, Tryptophan, Valine, Isoleucine, Leucine, Phenylalanine, Histidine, and Lysine are the essential amino acids Ø (Remember MATTVILPHLy). Peptide Ø Amino acids are linked together by formation of covalent bonds. Ø The covalent bond is formed between the -carboxyl group of one amino acid and the amino group of the next amino acid. Ø The bond so formed between the carboxyl and the amino groups, after elimination of a water molecule is called as a peptide bond and the compound formed is a peptide. Ø The peptide formed between two amino acids is a dipeptide; three amino acids is a tripeptide; few amino acids are an oligopeptide and many amino acids is a polypeptide. Ø In writing the peptide structure, the amino terminal (N-terminal) amino acid is written first and carboxyl terminal (C-terminal) amino acid written last. 30 Fundamental of Plant Biochemistry and Biotechnology AME 111 Ø Peptides of physiological interest Glutathione is a commonly occurring tripeptide (glutamyl cysteinyl glycine) in many living organisms. Ø It has a role in detoxification of toxic compounds in physiological system. Ø The nonapeptides (nine amino acids), oxytocin and vasopressin are important animal peptide hormones. Ø Oxytocin induces labor in pregnant women and controls contraction of uterine muscle. Ø Vasopressin plays a role in control of blood pressure by regulating the contraction of smooth muscles. Ø A dipeptide L-aspartyl-L-phenylalanine, is of commercial importance. This dipeptide is about 200 times sweeter than cane sugar. The methyl ester of this dipeptide is called as aspartame and marketed as an artificial sweetener for diabetics. 31 Fundamental of Plant Biochemistry and Biotechnology AME 111 Protein structure Protein are polymer of Amino acid , the structure of Protein is rather complex which can be divided into 4 levels of organization 1. Primary Structure 2. Secondary structure 3. Tertiary Structure 4. Quaternary Structure The structure of Protein is comparable with the structure of a building. The amino acids may be considered as the bricks, the wall as the Primary structure, the twists in a wall as the secondary structure, a full fledged self contained room as the tertiary structure. A building with similar and dissimilar rooms will be the quaternary structures. Primary Structure The primary structure of a protein refers to the linear sequence of amino acids in the polypeptide chain. The primary structure is held together by covalent bonds such as peptide bonds, which are made during the process of protein biosynthesis or translation. The two ends of the polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Counting of residues always starts at the N-terminal end (NH2 -group), which is the end where the amino group is not involved in a peptide bond. Secondary Structure The conformation of polypeptide chain by twisting or folding is referred to as secondary structure. The amino acid are located close to each other in their sequence. The most common type of secondary structure are the a) Alpha helix. b) Beta pleated sheet. Both Alpha helix and beta pleated sheet patterns are stabilized by hydrogen bonds between the carbonyl and N-H groups in the polypeptide’s backbone. 32 Fundamental of Plant Biochemistry and Biotechnology AME 111 Alpha Helix 1. The Alpha Helical structure was proposed by Pauling and Corey (1951) 2. The Alpha Helix is the most common spiral, rigid, rod like structure that forms when a polypeptide chain twists into a helical conformation. The screw sense of Alpha Helix can be right handed (right handed) or left handed (anticlockwise), however right handed helices are energetically more favorable. There are 3.6 amino acid residues per turn of the helix and the distance it rises per turn is 0.54 nm each residue is related to the next one by rise of 1.5 A. 3. The Alpha Helix is stabilize by extensive hydrogen bonds in between the N-H group of each amino acid and the carbonyl group of the amino acid four residue away, side chain of amino acids extended outward from the central helix. The hydrogen bond are individually weak but collectively they are strong enough to stabilize the Helix. 4. All the peptide bonds, except the first and the last in polypeptide chain participate in the H-bonding. Beta Pleated sheets This is the second type of structure, proposed by Pauling and Corey. 1. Beta pleated sheets form when two or more polypeptide chain segment line up side by side. Each individual segment is referred to as a beta-strand. Rather than being coiled, each Beta strand is fully extended. The distance between adjacent amino acids along a beta strand is approximately 3.5 A. 2. Beta pleated sheets are stabilized by hydrogen bonds that form between the polypeptide backbone N-H and carbonyl groups of adjacent strand, adjacent strand can be either parallel or antiparallel. 3. In parallel Beta pleated sheet structures, the polypeptide chains are arranged in the same direction. However in, antiparallel Beta pleated sheet chain run in opposite directions. Antiparallel beta pleated sheet are more stable than parallel beta pleated sheet because fully collinear hydrogen bonds form. 33 Fundamental of Plant Biochemistry and Biotechnology AME 111 Tertiary structure 1. The three dimensional arrangement of protein structure referred to a tertiary structure. 2. It is a compact structure with hydrophobic side chain held interior while the hydrophilic groups are on the surface of the protein molecule, this type of arrangement ensures stability of the molecule. 3. The following types of covalent and non-covalent interaction stabilize tertiary structure. i. Hydrophobic interaction ii. Electrostatic interaction iii. Hydrogen bond iv. Vander waal forces v. Covalent bonds. Quaternary structure 1. Some of the Proteins are composed of two or more polypeptide chains referred t o as subunits. The spatial arrangement of these subunits is known as Quaternary Structure. Subunit in multisubunit protein may be identical or quite different. Multisubunit Proteins in which some or all subunits are identical are referred to as Oligomer and identical units are referred to as protomers. 2. Polypeptide subunits assemble and are held together by the noncovalent interactions such as hydrophobic interactions, ionic bond and hydrogen bonds. 34 Fundamental of Plant Biochemistry and Biotechnology AME 111 35 Fundamental of Plant Biochemistry and Biotechnology AME 111 Denaturation and Renaturation of protein · Renaturation refers to the attainment of an original, regular three-dimensional functional protein after its denaturation. · When active pancreatic ribonuclease A is treated with 8M urea or mercaptoethanol, it is converted to an inactive, denatured molecule. · When urea or mercaptoethanol is removed, it attains its native (active) conformation. 36 Fundamental of Plant Biochemistry and Biotechnology AME 111 ENZYMES Enzyme are biocatalysts – the catalysts of life, a catalysts is defined as a substance that increases the velocity or rate of a chemical reaction without itself undergoing any change in the overall process. It is well known that highly complex synthetic and breakdown reactions take place much more rapidly and easily by the living organism. In the absence of the cell these chemical reactions would proceed too slowly. In the laboratory, hydrolysis of protein by a strong acid at 100 C takes at least a couple of days. The same protein is fully digested by the enzyme in gastrointestinal tract by the enzyme in gastrointestinal tract at body temperature (37 C) with in a couple of hours An enzyme is a protein that is synthesized in a living cell and catalyzes or speeds up a thermodynamically possible chemical reaction. The enzymes in on way modify the equilibrium constant (Keq) or the free energy change (G) of a reaction. Terminology Some of the terms used in enzymology are defined below: Substrate: The substrate acted upon by the enzyme. Product: The substrate formed as a result of the enzymatic action Active site or catalytic site: the site on the enzyme wherein the substrate is bound and is converted into products Regulatory site: The site other than the active site on the enzyme wherein the effector or modulator is bound and controls the rate of enzyme catalyzed reaction. Effector or modulator: The substance which binds at an allosteric site (the site other than the catalytic site) of the regulatory enzyme and may stimulate or inhibit the rate of enzyme catalyzed reaction Holoenzyme: A completely catalytically active enzyme. Holoenzyme = Apoenzyme + Cofactor (active) (inactive) (inactive) Cofactor: The non-protein component of the enzyme molecule required for complete activity. Cofactors can be classified into three groups; coenzymes, prosthetic groups and metal ions. 37 Fundamental of Plant Biochemistry and Biotechnology AME 111 Enzymes and their Cofactors Sl.no Co -factors Enzymes 1 Fe2+ or Fe3+ Cytochrome oxidase Catalase. Peroxidase. iv. Xanthine Oxidase. 2 Cu2+ Cytochrome oxidase Lysyl oxidase Superoxide Dismutase 3 Zn2+ Carbonic anhydrase Alcohal dehydrogenase Carboxy peptidase 4 Mg2+ Hexokinase Enolase Glucose 6 Phosphatase 5 Mn2+ Arginase Enolase Pyruvate carboxylase 6 K+ Pyruvate Kinase 7 Ni2+ Urease 8 Mo Dinitrogenase Xanthine oxidase 9 Se Glutathione peroxidase Apoenzyme: The protein component of the enzyme. Coenzyme: The non-protein organic molecule that is not covalently bound and can be readily dissociated from the protein component of the enzyme by dialysis. Prosthetic group: The non-protein component that is covalently bound and not readily dissociated from the protein component of the enzyme by dialysis. 38 Fundamental of Plant Biochemistry and Biotechnology AME 111 Zymogen or proenzyme : The precursor form of the enzyme in an inactive form. Activator: Any substance that increases the rate of an enzyme-catalyzed reaction. Inhibitor: Any substance that reduces or inhibits the rate of an enzyme- catalyzed reaction. Turnover number (molar activity): The number of molecules transformed into product (s) per minute by one molecule of the enzyme. Specific activity: The amount of enzyme units present per milligram of protein. Enzyme unit: The amount of enzyme which transforms one micromole of substrate per minute under defined assay conditions. Michaelis-Menten constants (Km): The substrate concentration at half the maximal velocity of the enzyme-catalyzed reaction. General characteristics The general characteristics of enzymes are as follows: Specialized proteins All the enzymes as far as known are specialized proteins that catalyze biochemical reactions. Enzymes show all the properties of protein, i.e., like proteins, enzymes are chemically made up of amino acids as building-blocks linked by peptide bonds; can be hydrolyzed to yield a mixture of constituent amino acids; lose catalytic activity if subjected to extreme pH, temperature, strong acids or bases, organic solvents or other conditions which denature protein and give typical color tests like biuret and FCR (Folin-Ciocalteu reagent) reactions. Biological organic catalysis Enzymes are also referred to as biological organic catalysis as: · Thy enhance the rate of specific chemical reactions. · They do not shift the eqauilibria of the reactions they catalyze. · They are regenerated during the course the reaction and · They are effective in concentrations that are minute as compared with the concentrations of substrates undergoing reaction. 39 Fundamental of Plant Biochemistry and Biotechnology AME 111 Fig: Activation energy How an enzyme enhances the rate of chemical reactions? For a chemical reaction A + B → C + D to occur, three criteria must be met: 1. The reactants, called substrates (A, B) must collide 2. The molecular collision must occur with the correct orientation, and 3. The reactants must have sufficient energy. This energy is called the activation energy which is the amount of energy required to bring all the molecules at a given temperature to the transition sate. More specifically, it is the difference in energy between the ground state of reactants and the transition state. Enzymes enhance the rate of chemical reaction by decreasing the activation energy of the reaction and with a high probability of correct orientation of reactants. The transition state refers to the state at which all molecules at any given instant possess enough energy to attain an activated condition before the reactants can be converted to 40 Fundamental of Plant Biochemistry and Biotechnology AME 111 products (C, D). This state is at the top of the energy barrier separating the reactants and products. In the normal course, not all molecules acquire sufficient energy to attain the transition state by collision. There are two ways of increasing the reaction rate by: a) Increasing the temperature which increases thermal motion and energy of molecules capable of entering the transition state, and b) Lowering the energy of the transition state by the addition of catalysts. In the enzyme-catalyzed reaction, the lowering of activation energy is achieved by the formation of activated ES (enzyme-substrate) complex by the combination of the enzyme with the substrate. This is clearly represented in Fig(6.1) where, (1) is the energy of activation, the difference in the energy level of reactants, A and B and the transition state for uncatalyzed reaction, (2) it is energy of activation for the formation of ES complex and is much less than (1) and (3), is the difference in energy levels between the reactant and product, i.e., overall free energy change of the reaction which remains the same in both catalyzed and uncatalyzed reactions. Specificity Unlike inorganic catalysts enzymes, the biological organic catalysts are more specific toward their substrates and for the type of reactions that catalyze. The term specificity refers to the affinity of the enzyme towards its substrate. Types of specificity Enzymes exhibit different types of specificity · Absolute specificity: Some enzymes act on only one substrate. Such enzymes are said to exhibit absolute specificity. For example, succinic dehydrogenase, a key enzyme of TCA cycle catalyzes only the oxidation of succinate to fumarate. · Absolute group specificity: Some other enzymes act on a very small group of substrates having the same functional group but at different rates. Such enzymes are said to exhibit absolute group specificity. For example, alcohol dehydrogenase oxidizes both ethanol and methanol which have common hydroxyl group. Similarly, hexokinase not only phosphorylats glucose but also fructose and mannose. 41 Fundamental of Plant Biochemistry and Biotechnology AME 111 · Relative group specificity: Some other enzymes exhibit relative group specificity. A given enzyme can act upon more than one group of substrates. For example, trypsin catalyzes the hydrolysis of both ester and amide bonds. · Stereospecificity: Many other enzymes show stereospecificity i.e., a given enzyme can act upon only particular stereoisomer. For example, L-amino acid oxidase acts only on L- amino acid but not on its D-form of amino acid. D-amino oxidase acts only on D-amino acid but not on its L-form. The enzymes are so specific since the active site of each enzyme has the proper shape, size and charge to bind certain substructure only and to catalyze the conversion of these substrates to specific products. Active site Enzyme –catalyzed reactions occur at an asymmetric pocket of the enzyme called the active site. The conformation and chemical composition of the active site determines the specificity of enzymatic catalysis. Theoretically, the active site can be subdivided a) A binding site, which includes the amino acid residues come into contact with the substrate, and (b) a catalytic site, which includes residues directly responsible for catalysis. However, the binding and bond-breaking processes are important in catalysis by enzymes. In all cases where conformations of enzymes have been determined by X-ray crystallography, the active site has been found to a relatively small area of the enzyme surface. Furthermore, the active site is a specific three – dimensional region having a unique arrangement of amino acid side chains, which are often contributed by amino acids situated quite far apart on the linear sequence of polypeptide chain. For example, the groups on the active site of enzyme lysozyme are contributed by the side chains of glutamate 35, aspartate – 52, tryptophan – 62 and 63 and aspartate – 101 (number after each amino acid refers to its location along the polypeptide chain). The rest of the polypeptide chain may be extremely important in maintaining the correct three – dimensional conformation of the active site. 42 Fundamental of Plant Biochemistry and Biotechnology AME 111 Theories for enzyme – substrate binding Two theories have been proposed to explain interaction of substrate and enzyme. 1. Lock and key model According to the lock and key model proposed by Emil Fisher in 1894, the substrate and enzyme have structural complementarily and fit together like lock and key i.e., the active site of the enzyme has a complementary shape of the substrate to form enzyme- substrate complex. This model has proved to be essentially correct in the case of enzymes known to exhibit absolute specificity. 2. Induced-fit theory This theory proposed by D.E.Koshland in 1968 suggests that the substrate binds at the active site of the enzyme and then modifies the shape of the active site so that it becomes complementary for the substrate binding. For example, binding of substrate to lysozyme takes place in this way. 43 Fundamental of Plant Biochemistry and Biotechnology AME 111 Nomenclature Naming of enzymes In the past enzymes were named in a haphazard manner as an when they were discovered. The ways in which enzymes are or have been named are listed below: 1. The first enzymes studied have been named for their colour their localization within the body or after the person who discovered them. However, this nomenclature had not been agreeable to many. 2. Later, many enzymes have been named by adding the suffix “ase” to the name of the substrate, for example, urease catalyzes the hydrolysis of arginine to ornithine and urea and so on. However, this nomenclature has not been always practicable. 3. Further, a systematic classification of enzymes has been adopted on the recommendation of an International Enzyme Commission as listed in the 1973 edition of Enzyme Nomenclature with a few exceptions. According to this, enzymes have been classified into six major classes and sets of classes based on the nature and type of reactions catalyzed. According to this, each enzyme is assigned: A recommended name: It is usually short and appropriate everyday use. A systematic name: It identifies the reaction the enzymes catalyzes and A classification number: It is used where accurate and unambiguous identification of an enzyme is required, as in research journals, abstracts and indexes. An example is given by the enzyme catalyzing the reaction: ATP + creatine → ADP + phosphocreatine The recommended name: creatine kinase The systematic name: ATP: creatine phosphotransferase Classification number : EC 2.7.3.2, where EC stands for Enzyme Commission, the first digit (2) for the major class name (transferases), the second digit (7) for the subclass 44 Fundamental of Plant Biochemistry and Biotechnology AME 111 (phosphotransferases), the third digit (3) for the sub-subclass (phosphotransferases with a nitrogenous group as acceptor) and the fourth digit (2) designates creatine kinase. Enzyme classification Enzymes can be classified into six major classes based on the nature and type of reactions catalyzed as given below: 1. Oxidoreductases: These enzymes catalyze oxidation or reduction reactions by transfer of hydrogen or electrons, e.g., succinic dehydrogenase. 2. Transferases: These enzymes are involved in transferring functional groups between donors and acceptors. The amino, acyl, phosphate, one-carbon and glycosyl groups are the major moieties that are transferred e.g., glutamic pyruvic transminase. 3. Hydrolases: This group of enzymes can be considered as special class of transferases in which the donor group is transferred to water. The generalized reaction involves the hydrolytic cleavage of C-O, C-N, O-P and C-S bonds. The cleavage of the peptide bond by peptidases is good example of this reaction. The proteolytic enzymes are a special class of hydrolases called peptidases. 4. Layses: These enzymes remove the groups of water, ammonia or CO2 from the substrate to cleave double bond or conversely, add these groups to double bonds e.g., furmarase. 5. Isomerases: These are a very heterogeneous group of enzymes that catalyze isomerizations (i.e., structural rearrangements within a molecule) of several types. These include cis-trans, keto-enol, and adose-ketose interconversion. Isomerases that catalyze inversion at asymmetric carbons are either epimerases or racemases. Mutases involve the intramulecular transfer of a group such as phosphoryl group. 6. Liagases (synthetases): These enzymes are involved in synthetic reactions where two molecules are joined together at the expense of breakdown of nucleoside-triphosphates. The formation of aminoacyl tRNAs, acetyl coenzyme A, glutamine, and the addition of CO2 to pyruvate are reactions catalyzed by ligases, e.g. pyruvic carboxylase. 45 Fundamental of Plant Biochemistry and Biotechnology AME 111 The six major classes of enzymes and the type of reactions catalyzed are summarized in Table below. Table: Classification of enzymes and types of reactions catalyzed S.N. Enzyme class Reactions catalyzed 1. Oxidoreductases Oxidation and reduction of substrates (usually involve hydrogen transfer) Dehydrogenases Transfer of hydrogen atoms from substrate to NAD* Oxidases Transfer of hydrogen atoms from substrate to oxygen Oxygenases Partial incorporation of oxygen to substrate Peroxidases Transfer of electrons from substrate to hydrogen peroxide 2. Transferases Transfer of a chemical group (such as a methyl group, amino group, phosphate group from one molecule to another Phosphorylases Addition of orthophosphate to substrate Transaminases Transfer of amino group from one substrate to another Kinases Transfer of phosphate from ATP to substrate 3. Hydrolases Cleavage of bonds by the addition of water Phosphatases Removal of phosphate from substrate Peptidases Cleavage of peptide bonds 4. Lyases Addition of groups to double bond (-C=C-, C=O, -C=N-) Decarboxylases Removal of carbon dioxide from substrate 5. Isomerases Rearrangement of atoms of a molecule 6. Liagases Formation of new bonds using energy from (simultaneous) breakdown of ATP Synthetases Joining two molecules together 46 Fundamental of Plant Biochemistry and Biotechnology AME 111 Factors affecting the rate of enzyme-catalyzed reactions Several factors are known to influence the rate of enzyme-catalyzed reaction, chief of them being : 1. Enzyme concentration 2. Substrate concentration 3. Temperature 4. pH and 5. Activators and inhibitor Effect of enzyme concentration With few exceptions, the initial velocity (V0 ) of enzymatic reactions bears a linear relationship with the concentration of the enzyme [E], provided other conditions do not act as limiting factors. If an excess substrate is present, doubling the enzyme concentration usually doubles the rate of formation of end products. This usually applies only at the start of the reaction, for the end-products of the reaction often have an inhibitory effect on the enzyme, and decrease its efficiency. Fig 6.5 is a graph which relates enzyme concentration to enzyme activity, all other factors being held constant. The dotted part is hypothetical, and almost impossible to attain in vitro; it may occur to a limited extent in the living cell. Effect of substrate concentration The concentration of substrate [S] affects the rate of the enzyme-catalyzed reaction. When the enzyme concentration is kept constant and the substrate concentration varied, the rate of enzyme-catalyzed reaction increases linearly upto a certain concentration of substrate after which a point is finally reached, where the enzyme is saturated with substrate. When this saturation point is reached, further increases in substrate concentration have no influence on rate of the enzyme-catalyzed reaction which is due to saturation of all the active sites of the enzyme by the substrate and partly due to more rapid accumulation of end-products causing inhibition of enzyme. The typical shape of curve obtained in this case is a nonlinear hyperbolic relationship between V0 and substrate concentration. 47 Fundamental of Plant Biochemistry and Biotechnology AME 111 Pioneering work on the kinetic studies of enzyme-catalyzed actions was made by A. Brown (1902) and V. Henri (1903). This was further developed by L. Michaelis and M.L. Menten in 1903 and by G.E. Briggs and J.B.S.Haldane in 1926. To account for hyperbolic relationship. Brown put forward hypothesis that the enzyme (E) reversibly combines with the substrate (S) to form an enzyme-substrate (ES) complex which is decomposed to yield product (P) and the free enzyme in its original from. Based on the work of Briggs and Henri, Michaelis and Menten derived a mathematical equation which is consistent with the empirical data represented They assumed that a rapid equilibrium exists between E, S and ES and that ES-complex breakdown of ES to E and S. This plot is based on the following equation for the behavior of a simple enzyme-catalyzed reaction in which one substrate is converted to one product. Where k+1, k-1 , k+2 and k-2 are the velocity constants for the above four steps. By rate measuring the of reaction, the rate constant k-2 may be ignored because not be initial present to make the reverse reaction proceed at significant rate. enough products will 48 Fundamental of Plant Biochemistry and Biotechnology AME 111 The equation they obtained is called Michaelis-Menten equation which is, Where Vo = Initial velocity of the enzymatic reaction Vmax = Maximal velocity at infinite substrate concentration, [S] = Substrate concentrtion and Km = Michaelis – Menten constant In Michaelis – Menten equation, when [S] = Km, then Or Vo = Vmax 2 49 Fundamental of Plant Biochemistry and Biotechnology AME 111 Hence, Km can be defined as the concentration of the substrate at which the reaction rate is half the maximal velocity. Usually, Km has dimension moles per liter and its independent of enzyme concentration. Determination of Km and Vmax The hyperbolic curve of the Vo verses [S] plot does not permit the determination of exact values of Km and Vmax. This limitation can be overcome by plotting the same kinetic data in different ways. The most popular approach is simple by taking reciprocal of both sides of Michaelis- Menten equation to yield a straight-line rate equation, called a Lineweaver-Burk equation which is, Fig. Lineweaver–Burk plot 50 Fundamental of Plant Biochemistry and Biotechnology AME 111 By plotting l/Vo versus l/[S], a straight line is obtained having a slope of Km/Vmax, an intercept of l/Vmax on l/vo axis and an intercept of –l/Km and l/[S] axis. Such a plot is called a Lineweaver- Burk plot or double reciprocal plot (Fig. 6.7 ). By determining the value of l/Vmax, Km can be determined. Km can also be directly determined by extrapolation of the line to meet the l/(S) axis where the intercept is –l/Km. The double-reciprocal plot has the advantage that measurement of the velocity of the reaction can be made at a number of substrate concentrations and then extrapolated back to infinite substrate concentrations at the intercept, which gives a straight-line so that accurate values of Vmax and Km can be determined. This also gives variable information on enzyme inhibition. Significance of Km and Vmax The Km value which is expressed in molar concentration is characteristic of each individual enzyme. Km is not a fixed value may vary with structure of substrate, pH and temperature. For enzyme having more than one substrate, each substrate has a characteristic Km value. Km helps to evaluate the specificity of action of given enzyme towards similar substrates. The substrate with the lowest Km value has highest apparent affinity for the enzyme. It also establishes an approximate value for the intercellular level of substrate as the enzymes are not necessarily saturated with their substrates. Knowledge of the Km of an enzyme can be of consideration value in investigations of metabolic control. Vmax represents the efficiency of enzyme action and can be used to compare the catalytic efficiency of different enzymes. 51 Fundamental of Plant Biochemistry and Biotechnology AME 111 Effect of temperature As with most chemical reactions, the rate of an enzyme-catalyzed reaction increases with temperature, however, in the case enzymes there is a temperature at which thermal denaturation of protein causes a loss of activity and the rate begins to slow down. For each enzyme there is an optimum temperature at which it is more active and its activity slows down when the temperature is changed in either direction away from this optimum. This is because, temperature changes cause slight changes in the three-dimensional shape of protein, increases in temperature may cause the protein to loosen up and become less compact. Even small changes in the dimensions of its active site can make an enzyme a less efficient catalyst. The velocity of a chemical reaction is related to temperature according to Vant Hoff’s law which states that a rise of 10ºC will double the speed of a chemical reaction. Velocity at (T+10º) ------------------------ Velocity at Tº The temperature coefficient is usually expressed by the symbol, Q10 which is also applicable to enzyme-catalyzed reactions. For most enzyme-catalyzed reactions. Q10 is approximately 2 at lower temperatures, but gradually drops off until the rate is 1 (or lower) at higher temperatures. Fig.6.8 shows the general effect of temperatures (bell shaped curve) on most enzyme-catalyzed reactions. Most enzymes show a temperature optimum between 25 and 37ºC but are inactivated at temperatures above 55ºC although enzymes in thermophilic bacteria are active even at temperature exceeding 55ºC. Effect of pH The activity of the enzyme is markedly influenced by the pH of the medium in which the reaction occurs. It is usually found that activity is shown over a limited range of pH and within this range, a bell-shaped activity curve (Fig ) is often observed when enzyme activity is plotted against pH. Every enzyme has a characteristic pH optimum wherein the activity is at maximum and the activity decreases on either side of this value. Most enzymes have pH 52 Fundamental of Plant Biochemistry and Biotechnology AME 111 optimum in the region 6-8, which, however, varies with the source of the enzyme, the kind of substrate, the kind of buffer and the temperature. For example, pepsin has a pH optimum around 2, for enzymes of plants and fungi it is 4.0-6.5 and for most enzymes of higher animals it is 6.5-8.0. At extreme pH values protein denaturation will occur with concomitant loss of enzyme activity. Slight changes in pH which do not denature the protein, may alter the state or degree of ion