metabolic pathways and the origin of secondary metabolites

Chapter 18 Basic metabolic pathways and the origin of secondary metabolites



ENZYMES 148

PHOTOSYNTHESIS 152

CARBOHYDRATE UTILIZATION 153

GLYCOSIDES 154

FATS AND FATTY ACIDS 155

AROMATIC BIOSYNTHESIS 156

AMINO ACIDS 158

PEPTIDES AND PROTEINS 161

ISOPRENOID COMPOUNDS 162

SECONDARY METABOLITES 165

The biosynthesis of both primary and secondary metabolites is dependent on the highly organized structure of the plant and animal cell. Unlike animal cells, those of plants possess a rigid cell wall and are separated one from another by an intercellular structure, the middle lamella. Direct connection between adjacent cells is maintained by primary pit fields through which pass the plasmodesmata. Within the cell wall is the protoplast consisting of cytoplasm, nucleus and various organelles.


The light microscope shows the nucleus to contain various inclusions such as nucleoli, chromosomes (stainable during cell division) and the nuclear sap. The nucleus appears to be suspended within the cell by the cytoplasm, in which there may be large vacuoles with their own characteristic contents (crystals, aleurone grains, etc.). Other cytoplasmic inclusions are mitochondria, Golgi bodies, lysosomes and plastids (chloroplasts, chromoplasts, leucoplasts), but their structure is not resolvable, because of their small size. Electron microscopy shows a number of the subcellular organelles to have a highly organized fine structure suited to the many and varied biochemical processes which they perform.


Although, because of varied form and function, it is not possible to illustrate a ‘typical’ plant cell Fig. 18.1A shows diagrammatically the structures that might be expected in an unspecialized young root cell. Such a cell possesses a rigid wall, which immediately distinguishes it from an animal cell, but no chloroplasts and only small vacuoles are present. In a green plant cell (Fig. 18.1B) the same components are present but the large vacuole has oppressed the nucleus and cytoplasm towards the wall; green plastids often with starch granules are common.


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Fig. 18.1 The plant cell. A, diagrammatic representation of an undifferentiated cell: c, cytoplasm; e.r, endoplasmic reticulum; g, Golgi apparatus; l, lysosome; m, mitochondrion; m.l, middle lamella; n, nucleus; nl, nucleolus; n.m, nuclear membrane; p, pit in wall; r, ribosomes, w, primary cell wall; v, vacuole. B, green plant cell: cp, chloroplast; m, mitochondrion, n, nucleus; v, vacuole. C, Flow chart of some cell metabolites.


The organelles allow the creation of different chemical environments within one cell, and furthermore, by their structure they increase the area available for surface reactions which are all-important in biological systems. A description of the various organelles is given in the 14th edition of this book and Fig. 18.1C illustrates some aspects of their interdependence in the normal functioning of the cell. The molecular structures of these bodies have been extensively studied and details will be found in standard botanical texts.


Some basic metabolic pathways appear to be similar in both plants and animals, whereas others are more restricted in their occurrence. It is to the secondary plant products (i.e. those not necessarily involved in the essential metabolism of the cell) that the majority of vegetable drugs owe their therapeutic activity and so it is in these that pharmacognosists are particularly interested. However, as illustrated in Fig. 18.2, the production of these secondary metabolites is dependent on the fundamental metabolic cycles of the living tissue and so a brief indication of the latter will also be given; for fuller accounts of these, the student should consult a standard work on plant biochemistry.


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Fig. 18.2 Origins of some secondary metabolites in relation to the basic metabolic pathways of plants. DOX=deoxyxylulose; IPP=isopentenyl diphosphate; MVA=mevalonic acid.



ENZYMES


Many reactions occurring in the cell are enzyme-dependent, and before anything was known of the chemical nature of these substances it was recognized that they were organic catalysts produced by animal and vegetable cells. Their wide distribution and the delicacy of their operation has long been appreciated; they engineer reactions at normal temperatures and at pH values around neutral in a manner not possible in the laboratory.


An enzyme usually acts on one substance or class of substances, since it is specific for a particular atomic group or linkage. Specificity, however, varies; lipases are, in general, not highly specific, whereas fumarase acts only upon L-malate and fumarate, while D-malate is a competitive inhibitor of fumarase. Enzymes are also stereo- and regio- specific in their actions and, as it becomes possible to prepare more rare examples, organic chemists are becoming increasingly aware of enzyme potential for carrying out single-step transformations with complete stereochemical exactitude, an aspect important in the synthesis of many drugs. An enzyme will convert many thousand times its own weight, and the gradual diminution in activity which takes place is probably due to secondary reactions which bring about destruction of the enzyme.


The enzymology of the secondary metabolic pathways in plants has still been little investigated but progress is being made and here again cell cultures have proved useful in that they are often a better source for the isolation of enzymes than is the differentiated plant. In some cases, e.g. cell cultures of volatile oil-containing plants, little or no oil accumulates in the culture owing to the absence of storage receptacles but the relevant enzymes for terpenoid synthesis are still manufactured and preparations of them can be made.


New horizons for the study of the enzymology of secondary metabolism have now opened up as a result of advances in gene technology. In suitable instances, by cloning, the cDNA responsible for an enzyme’s synthesis can be expressed in another organism such as a bacterium and large amounts of enzyme prepared. By conventional methods only very small amounts of purified enzyme could be obtained from the original plant material. Of particular interest has been the isolation, characterization and cloning of the enzyme strictosidine synthase. This governs the key reaction for the commencement of the biosynthesis of the very many monoterpenoid indole alkaloids, namely the condensation of tryptamine and secologanin to give 3alpha;(S)-strictosidine (see Chapter 26); for a review (126 references) on this enzyme see T. M. Kutchan, Phytochemistry, 1993, 32, 493.


By means of relatively new technology, enzymes can be immobilized on a suitable carrier either in whole plant cells or as the isolated enzyme. In this way these biocatalysts can be repeatedly used in analytical and clinical chemistry, or to effect specific chemical transformations.


Like other catalysts, enzymes influence the rate of a reaction without changing the point of equilibrium. For example, lipase catalyses either the synthesis of glycerides from glycerol and fatty acids or the hydrolysis of glycerides, the final point of equilibrium being the same in either case. Similarly, β-glucosidase (prunase) has been used for both the synthesis and the hydrolysis of β-glucosides. In plants such reversible reactions may proceed in one direction or the other under different conditions, often resulting in daily and seasonal variations in the accumulation of metabolites.


Enzymes are colloidal in nature and consist of protein or contain protein as an essential part. They may be partly purified, and in some cases isolated, by the methods of protein chemistry (i.e. by fractional precipitation, dialysis and, more recently, gel and affinity chromatography, see Chapter 17). Most enzymes are soluble either in water or in dilute salt solutions and are precipitated by alcohol or acetone (acetone powders) and by high concentrations of salts. They are inactivated by heat, ultraviolet light and X-rays or by any treatment which brings about denaturation of proteins.


The activity of enzymes is markedly affected by the reaction of the medium and the presence of substances such as salts. It is well known, for example, that pepsin works only in an acid medium and trypsin in an alkaline one. In general, carbohydrases have pH optima of 3.8–7.5 and lipases optima of pH 5–8, while enzymes which act on bases all have optima more alkaline than pH 7.


The effect of heat on enzymes is of considerable importance in the drying of drugs. At low temperatures enzymic changes are not usually marked, although the proteolytic or protein-splitting enzymes in cod livers do bring about some hydrolysis at temperatures approaching zero. The optimum working temperatures of different enzymes vary, but they usually lie between 35 and 50 °C. At temperatures of about 60 °C destruction of the enzymes is usually fairly rapid, although considerable loss may take place below this temperature. When dry, enzymes show increased resistance to heat; thus, zymase, which in the presence of moisture is rapidly inactivated at 50 °C, will, when dry, resist a temperature of 85 °C.



Chemical nature


Following the isolation of urease in crystalline form by Sumner in 1926, many other enzymes have been prepared in a crystalline form and


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their protein nature has been established. Their molecular weights are high, but vary from about 9000 (hydrogenase) to about 1 000 000. In many cases the component amino acids are known and in some cases (e.g. ribonuclease) the amino acid sequence within the molecule has been determined. During the isolation of an enzyme, the purified protein (apoenzyme) may be inactive but regains its activity in the presence of an essential coenzyme or activator, which may be organic or inorganic.



Coenzymes


Some enzymes which require the presence of smaller organic molecules, called coenzymes, before they can function are of very common occurrence and participate in a large number of important biochemical reactions. One group of coenzymes consists of esters of phosphoric acid and various nucleosides. The adenosine and uridine phosphates contain one basic unit each (mononucleotides); they serve to transport energy in the form of high-energy phosphate bonds and this energy is made available for biochemical reactions in the presence of the appropriate enzyme by hydrolysis of the bond. Thus, the terminal phosphate bond of the adenosine triphosphate (ATP) on hydrolysis to adenosine diphosphate (ADP) affords 50 000 J mol−1.


Uridine triphosphate (UTP) is involved in the synthesis of sucrose via diphosphate glucose which is also associated with the formation of uronic acids and cellulose.


Nicotinamide-adenine dinucleotide (NAD) and nicotinamide-adenine dinucleotide phosphate (NADP) contain two basic units each and are termed dinucleotides. They function in oxidation–reduction systems with appropriate enzymes; the oxidized forms are written NAD+ and NADP+ and the reduced forms NADH and NADPH respectively.



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Another important coenzyme is coenzyme A (CoA), which contains the units adenosine-3,5-diphosphate, pantothenic acid-4-phosphate and thioethanolamine. It participates in the transfer of acetyl and acyl groups, acetyl-CoA (active acetate) having a central role in plant and animal metabolism.



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Riboflavine (Fig. 31.2) is a component of the two coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). They participate in the biological oxidation–reduction system, and FAD facilitates the transfer of H+ ions from NADH to the oxidized cytochrome system.


Other coenzymes are the decarboxylation coenzymes thiamine, biotin and pyridoxine (Fig. 31.2). Folic acid (Fig. 31.2) derivatives participate in enzymatic reactions which involve one-carbon fragment transfers.


A series of quinones e.g. plastoquinone and ubiquinone are widely distributed in plants, animals and microorganisms and function in biological electron transfer processes.



Classification of enzymes


As more enzymes are isolated, it becomes important to have a precise scheme of classification and nomenclature. Many long-established names such as pepsin, prunase, diastase and names for enzyme mixtures, such as emulsin and zymase, continue in use. A step towards uniform nomenclature was made when they were denoted by the name of the substrate and the termination ‘-ase’. Classes of enzymes were named similarly. Thus, the general term ‘esterase’ includes lipases, which hydrolyse fats; chlorophyllase hydrolyses chlorophyll; etc. The 1961 Report of the Commission on Enzymes*


* This report has a numbered classification for each enzyme. For example, the enzyme present in garlic, alliine-lyase, is numbered 4.4.1.4. The first number denotes the fourth of the six main groups already mentioned and the other numbers further subdivisions. Thus:


4 = lyases

4.4 = carbon-sulphur lyases

4.4.1 = In this example a further subdivision is not required.

4.4.1.4 = the fourth enzyme listed in the group, namely alliine-lyase (formerly known as alliinase) or allyl sulphinate-lyase.

made the recommendation that the chemical reactions catalysed be generally adopted for classification and nomenclature. This, of course, presupposes that the exact chemical reaction is known; see also the 1984 International Union of Biochemistry publication (London, UK: Academic Press) Enzyme Nomenclature and any later reports. Enzymes are classified into six main groups (Table 18.1).


Table 18.1 Classification of some enzymes.
























































Group Trivial name Systematic name
1. Oxidoreductases Glucose dehydrogenase β-D-Glucose: NAD(P)-oxidoreductase
p-Diphenyl oxidase (lactase) p-Diphenol: O2 oxidoreductase
Peroxidase Donor: H2O2 oxidoreductase
Catalase H2O2: H2O2 orthoreductase
2. Transferase α-Glucan phosphorylase α-1,4-Glucan: orthophosphate glucosyl-transferase
3. Hydrolases Lipase Glycerol ester hydrolase
Chlorophyllase Chlorophyll chlorophyllidohydrolyase
Tannase Tannin acyl-hydrolase
α-Amylase α-1,4-Glucan 4-glucano-hydrolyase
Inulase Inulin 1-fructanohydrolase
4. Lyases Aldolyase Ketose-l-phosphate aldehydelyase
  Decarboxylase L-Tryptophan-decarboxylase
5. Isomerases Maleate isomerase Maleate cis-trans-isomerase
6. Ligases (Synthetases) Asparagine synthetase L-Aspartate: ammonia ligase (ADP)


Oxidoreductases


As any oxidation implies a simultaneous reduction, the names ‘oxidase’ and ‘reductase’ may be applied to a single type of enzyme. Eleven different groups of oxidoreductases are recognized. Many oxidases (that is, enzymes that utilize molecular oxygen as acceptor) convert phenolic substances to quinones. They act on guaiacum resin to produce a blue colour which is used as a test for their detection. Oxidases include laccase (1.10.3.2), present in lac, and ascorbate oxidase (1.10.3.3), which is widely distributed in plants. Oxalate oxidase (1.2.3.4), a flavoprotein present in mosses and the leaves of higher plants, oxidizes oxalic acid into carbon dioxide and hydrogen peroxide. Peroxidases (1.11) are distinguished by the fact that they use hydrogen peroxide and not oxygen as the hydrogen acceptor. Catalase (1.11) must be regarded as an exception, since it catalyses the decomposition of hydrogen peroxide. An oxidoreductase of the morphine biosynthetic pathway (Chapter 26) has recently been purified and characterized; it catalyses the stereoselective reduction of salutaridine to 7(S)-salutaridinol using NADPH as cosubstrate.



Hydrolases


These include many different types, of which the following are some of pharmaceutical importance.


1 Hydrolysing esters. These include lipases (3.1), which may be of vegetable or animal origin and which hydrolyse glycerides.Mammalian lipases also hydrolyse other esters, such as phenyl salicylate, acetylcholine and atropine. Other esterases are chlorophyllase (3.1.1.14), which hydrolyses chlorophyll, and tannase (3.1.1.20), which hydrolyses ester links in tannins. Other esterases probably occur in many drugs which contain esters in their volatile oils (e.g. valerian).

2 Hydrolysing sugars and glycosides. To the important group of glycoside hydrolases (3.2.1) belong all those enzymes that hydrolyse sugars, carbohydrates and glycosides. Those hydrolysing sugars include β-fructofuranosidase (sucrase or invertase), lactase, maltase, gentiobiase and trehalase. Polysaccharide enzymes are represented by α-amylase (3.2.1.1), β-amylase, cellulase, lichenase, inulase. Among the glycoside-hydrolysing enzymes are β-glucosidase or β-D-glucoside glucohydralase (3.2.1.20), which has a wide specificity for β-D-glucopyranosides. More specific glucoside- hydrolysing enzymes are those acting on salicin, amygdalin, sinigrin and cardiac glycosides.

3 Hydrolysing the C–N linkage. Many enzymes act on peptide bonds. To this group belong the well-known animal enzymes pepsin, rennin, trypsin, thrombin and plasmin, and the vegetable enzymes papain (from Carica papaya) and ficin (from species of fig). All the above belong to the group 3.4.4. Other enzymes acting on linear amides (3.5.1) are asparaginase (present in liquorice and many other plants) and urease. Cyclic amides are acted on by penicillinase (3.5.2.6).


Lyases


Two important decarboxylases in the biosynthesis of monoterpenoid indole alkaloids and benzylisoquinoline alkaloids are L-tryptophan-decarboxylase (EC 4.1.1.27) and L-tyrosine/L-dopa-decarboxylase (EC 4.1.1.25), respectively.



Isomerases


Two enzymes important in pathways leading to medicinally important metabolites are isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2) amd chalcone isomerase (EC 5.5.1.6). The former is involved in the isomerization of IPP and dimethylallyl diphosphate (Fig. 18.18), an essential step in the formation of terpenoids, and the latter is a key enzyme catalysing the isomerization of chalcones to their corresponding flavanones (Fig. 18.11).



PHOTOSYNTHESIS


Photosynthesis, by which the carbon dioxide of the atmosphere is converted into sugars by the green plant, is one of the fundamental cycles on which life on Earth, as we know it, depends. Until 1940, when investigations involving isotopes were undertaken, the detailed mechanism of this ‘carbon reduction’ was unknown, although the basic overall reaction,



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had been accepted for many years.


Photosynthesis occurs in the chloroplasts: green, disc-shaped organelles of the cytoplasm which are bounded by a definite membrane and which are autoreproductive. Separated from the rest of the cell, the chloroplasts can carry out the complete process of photosynthesis. Bodies with similar properties are found in cells of the red algae but these contain, in addition to chlorophyll as the principal pigment, other tetrapyrrole derivatives—the phycobilins.


The light microscope reveals no definite internal structure of the chloroplasts, but electron microscopy shows these bodies to have a highly organized structure in which the chlorophyll molecules are arranged within orderly structures (grana), each granum being connected with others by a network of fibres or membranes. According to one theory, the flat chlorophyll molecules themselves are orientated between layers of protein and lipid molecules so that the whole chloroplast can be looked upon as a battery containing several cells (the grana), each cell possessing layers of plates (the chlorophyll molecules).


Two fundamental processes which take place in photosynthesis, both of which require light, are the production of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and phosphate and the light-energized decomposition of water (the Hill reaction; named after the English biochemist Robert Hill, 1899–1991):



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ATP is a coenzyme and the high energy of the terminal phosphate bond is available to the organism for the supply of the energy necessary for endergonic reactions. The Hill reaction produces free oxygen and hydrogen ions which bring about the conversion of the electron carrier, NADP, to its reduced form NADPH (see ‘Coenzymes’).


In this complicated process, two systems, Photosystem I and Photosystem II (also known as pigment systems I and II) are commonly referred to; they involve two chlorophyll complexes which absorb light at different wavelengths (above and below λ = 685 nm). Photosystem II produces ATP and Photosystem I supplies all the reduced NADP and some ATP. In these light reactions the chlorophyll molecule captures solar energy and electrons become excited and move to higher energy levels; on returning to the normal low-energy state, the electrons give up their excess energy, which is passed through a series of carriers (including in the case of Photosystem II plastoquinone and several cytochromes) to generate ATP. Photosystem I involves an electron acceptor and the subsequent reduction of ferredoxin in the production of NADPH. Reference to the current literature indicates that the nature and organization of the photosystems remains a very active research area. Students will have observed that an alcoholic solution of chlorophyll possesses, in sunlight, a red fluorescence—no carriers are available to utilize the captured energy and it is re-emitted as light. Hill first demonstrated in 1937 that isolated chloroplasts, when exposed to light, were capable of producing oxygen, provided that a suitable hydrogen acceptor was present. Work with isotopes has since proved that the oxygen liberated during photosynthesis is derived from water and not from carbon dioxide.


Following the light reactions, a series of dark reactions then utilize NADPH in the reduction of carbon dioxide to carbohydrate.


Current research suggests that terrestrial plants can be classified as C3, C4, intermediate C3–C4 and CAM plants in relation to photosynthesis.



C3 plants


The elucidation of the carbon reduction cycle (Fig. 18.3), largely by Calvin and his colleagues, was in large measure determined by methods dependent on exposing living plants (Chlorella) to 14C-labelled carbon dioxide for precise periods of time, some very short and amounting to a fraction of a second. The radioactive compounds produced were then isolated and identified. In this way a sequence for the formation of compounds was obtained. 3-Phosphoglyceric acid, a C3 compound, was the compound first formed in a labelled condition but it was only later in the investigation, after a number of 4-, 5-, 6- and 7-carbon systems had been isolated, that ribulose-1,5-diphosphate was shown to be the molecule with which carbon dioxide first reacts to give two molecules of phosphoglyceric acid. An unstable intermediate in this reaction is 2-carboxy-3-ketopentinol.


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Fig. 18.3 The path of carbon in photosynthesis (after Calvin); the formulae of the sugars involved are given in Chapter 20.

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