Major biochemicals and enzymes in metabolic biochemistry 9788132337836, 8132337832, 1283499274, 9781283499279

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First Edition, 2012

ISBN 978-81-323-3783-6

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Published by: University Publications 4735/22 Prakashdeep Bldg, Ansari Road, Darya Ganj, Delhi - 110002 Email: [email protected] 

Table of Contents Chapter 1 - Glucose Chapter 2 - Fructose Chapter 3 - Glycogen Chapter 4 - Starch Chapter 5 - Pyruvic Acid Chapter 6 - Urea Chapter 7 - Adenosine Triphosphate Chapter 8 - Other Biochemicals and Enzymes in Metabolic Biochemistry

Chapter- 1

Glucose

D-glucose

D-glucopyranose

Chain form

Ball and stick model

Fischer projection of the chain form Preferred IUPAC name Dextrose Systematic name (2R,3S,4R,5R)-2,3,5,4,6-Pentahydroxyhexanal Other names Blood sugar Corn sugar D-Glucose

Abbreviations CAS number PubChem ChemSpider UNII

Grape sugar Identifiers Glc 50-99-7 5793 5589 5SL0G7R0OK

EC number KEGG MeSH ChEBI ChEMBL RTECS number ATC code Beilstein Reference Gmelin Reference 3DMet

200-075-1 C00031 Glucose CHEBI:4167 CHEMBL1216 LZ6600000 B05CX01,V04CA02, V06DC01 1281604 83256 B04623 Properties Molecular formula C6H12O6 Molar mass 180.16 g/mol Exact mass 180.063388 Density 1.54 g/cm3 α-D-glucose: 146 °C Melting point β-D-glucose: 150 °C Solubility in water 91 g/100 ml (25 °C) Solubility in methanol 0.037 M Solubility in ethanol 0.006 M Solubility in tetrahydrofuran 0.016 M Thermochemistry Std enthalpy of −1271 kJ/mol formation ΔfHo298 Std enthalpy of −2805 kJ/mol combustion ΔcHo298 Standard molar 209.2 J K−1 mol−1 entropy So298 Hazards MSDS ICSC 0865

D-glucose or dextrose or grape sugar is the dextro-isomer of glucose (C6H12O6). The molecule is the mirror-image of L-glucose. D-glucose is a simple sugar (monosaccharide) and an important carbohydrate in biology. Cells use it as a source of energy and a metabolic intermediate. Glucose is one of the main products of photosynthesis and starts cellular respiration. Starch and cellulose are polymers derived from the dehydration of glucose. The name "glucose" comes from the Greek word glukus (γλυκύς), meaning "sweet". The suffix "-ose" denotes a sugar. Glucose exists in several different structures, but all of these structures can be divided into two families of mirror-images (stereoisomers). Only one set of these isomers exists in nature, those derived from the "right-handed form" of glucose, denoted D-glucose. Dglucose is often referred to as dextrose. The term dextrose is derived from dextrorotatory glucose. Solutions of dextrose rotate polarized light to the right (in Latin: dexter = "right" ).

Structure Although it is called a "simple sugar" (meaning that it is a monosaccharide), glucose is a complicated molecule because it adopts several different structures. These structures are usually discussed in the context of the acyclic isomer, which exists in only minor amounts in solution. Glucose is derived from hexanal, a chain of six carbon atoms terminating with an aldehyde group. The other five carbon atoms each bear alcohol groups. Glucose is called an aldohexose. In solution, glucose mainly exists as the six-membered ring containing a hemiacetal group, which arises from the reaction of the hydroxy group at C-5 and the aldehyde at C-1. Containing five carbon atoms and one oxygen atom, this ring is a derivative of pyran. This cyclic form of glucose is called a glucopyranose, of which two isomers exist. The asymmetric center at C-1, the site of the hemiacetal, is called the anomeric carbon atom. The ring closing process can give rise to two isomers, called anomers, which are labeled α-glucose and β-glucose. These anomers differ in terms of the relative positioning of the hydroxyl group linked to C-1. When D-glucose is drawn as a Haworth projection or in the standard chain conformation, the designation α means that the hydroxyl group attached to C-1 is positioned trans to the -CH2OH group at C-5, while β means that it is cis. An inaccurate but superficially attractive alternative method of distinguishing α from β is observing whether the C-1 hydroxyl is below or above the plane of the ring; this may fail if the glucose ring is drawn upside down or in an alternative chair conformation. The α and β forms interconvert over a timescale of hours in aqueous solution, to a final stable ratio of α:β 36:64, in a process called mutarotation. The ratio would be α:β 11:89 if it were not for the influence of the anomeric effect.

Isomers Aldohexoses have four chiral centers in their acyclic forms (i.e. ignoring the anomeric carbon). Four chiral centers give rise to 24 = 16 stereoisomers. These stereoisomers are classified into two classes with eight sugars in each, which are mirror images of each other. One class is labeled L and the other D. Only seven of these isomers are found in nature, of which D-glucose (Glu), D-galactose (Gal) and D-mannose (Man) are the most important. These eight isomers (including glucose itself) are diastereoisomers and belong to the D series.

The Fischer projection of the chain form of D-glucose

The chain form of D-glucose

α-Dglucopyranose

β-Dglucopyranose

Chain form: ball-and-stick model

Chain form: space-filling model

α-Dglucopyranose

β-Dglucopyranose

Physical properties All forms of glucose are colorless and soluble in water. Depending on conditions, three major forms can be crystallised: α-glucose and β-glucose, and the hydrated β-glucose.

Production

Glucose tablets

Biosynthesis In plants and some prokaryotes, glucose is a product of photosynthesis. In animals and fungi, glucose results from the breakdown of glycogen, a process known as glycogenolysis. In plants the breakdown substrate is starch. In animals, glucose is synthesized in the liver and kidneys from non-carbohydrate intermediates, such as pyruvate and glycerol, by a process known as gluconeogenesis. In some deep-sea bacteria glucose is produced by chemosynthesis.

Commercial Glucose is produced commercially via the enzymatic hydrolysis of starch. Many crops can be used as the source of starch. Maize, rice, wheat, cassava, corn husk and sago are all used in various parts of the world. In the United States, cornstarch (from maize) is used almost exclusively. Most commercial glucose occurs as a component of invert

sugar, an approximately 1:1 mixture of glucose and fructose. In principle, cellulose could be hydrolysed to glucose, but this process is not yet commercially practical.

Function

Glucose metabolism and various forms of it in the process. -Glucose-containing compounds and isomeric forms are digested and taken up by the body in the intestines, including starch, glycogen, disaccharides and monosaccharides. -Glucose is stored in mainly the liver and muscles as glycogen. -It is distributed and utilized in tissues as free glucose. Scientists can speculate on the reasons why glucose, and not another monosaccharide such as fructose, is so widely used in organisms. One reason might be that glucose has a lower tendency, relative to other hexose sugars, to react non-specifically with the amino groups of proteins. This reaction (glycation) reduces or destroys the function of many

enzymes. The low rate of glycation is due to glucose's preference for the less reactive cyclic isomer. Nevertheless, many of the long-term complications of diabetes (e.g., blindness, renal failure, and peripheral neuropathy) are probably due to the glycation of proteins or lipids. In contrast, enzyme-regulated addition of glucose to proteins by glycosylation is often essential to their function.

As an energy source Glucose is a ubiquitous fuel in biology. It is used as an energy source in most organisms, from bacteria to humans. Use of glucose may be by either aerobic respiration, anaerobic respiration, or fermentation. Carbohydrates are the human body's key source of energy, through aerobic respiration, providing approximately 3.75 kilocalories (16 kilojoules) of food energy per gram. Breakdown of carbohydrates (e.g. starch) yields mono- and disaccharides, most of which is glucose. Through glycolysis and later in the reactions of the citric acid cycle (TCAC), glucose is oxidized to eventually form CO2 and water, yielding energy sources, mostly in the form of ATP. The insulin reaction, and other mechanisms, regulate the concentration of glucose in the blood. A high fasting blood sugar level is an indication of prediabetic and diabetic conditions. Glucose is a primary source of energy for the brain, and hence its availability influences psychological processes. When glucose is low, psychological processes requiring mental effort (e.g., self-control, effortful decision-making) are impaired. α-D-Glucose

Hexokinase ATP

α-D-Glucose-6-phosphate

ADP

Compound C00031 at KEGG Pathway Database. Enzyme 2.7.1.1 at KEGG Pathway Database. Compound C00668 at KEGG Pathway Database. Reaction R01786 at KEGG Pathway Database.

Glucose in glycolysis Use of glucose as an energy source in cells is via aerobic or anaerobic respiration. Both of these start with the early steps of the glycolysis metabolic pathway. The first step of this is the phosphorylation of glucose by hexokinase to prepare it for later breakdown to provide energy. The major reason for the immediate phosphorylation of glucose by a hexokinase is to prevent diffusion out of the cell. The phosphorylation adds a charged

phosphate group so the glucose 6-phosphate cannot easily cross the cell membrane. Irreversible first steps of a metabolic pathway are common for regulatory purposes. In anaerobic respiration one glucose molecule produces a net gain of 2 ATP molecules (4 ATP molecules are produced during glycolysis but 2 are required by enzymes used during the process) . In aerobic respiration a molecule of glucose is much more profitable in that a net worth of 32 ATP molecules are generated (34 gross with 2 being required in the process) .

As a precursor Glucose is critical in the production of proteins and in lipid metabolism. In plants and most animals, it is also a precursor for vitamin C (ascorbic acid) production. It is modified for use in these processes by the glycolysis pathway. Glucose is used as a precursor for the synthesis of several important substances. Starch, cellulose, and glycogen ("animal starch") are common glucose polymers (polysaccharides). Lactose, the predominant sugar in milk, is a glucose-galactose disaccharide. In sucrose, another important disaccharide, glucose is joined to fructose. These synthesis processes also rely on the phosphorylation of glucose through the first step of glycolysis.

Glucose for use in the laboratory. Industrial use In industry, glucose is used as a precursor to make vitamin C in the Reichstein process, to make citric acid, gluconic acid, bio-ethanol, polylactic acid, sorbitol.

Sources and absorption Most dietary carbohydrates contain glucose, either as their only building block, as in starch and glycogen, or together with another monosaccharide, as in sucrose and lactose.

In the lumen of the duodenum and small intestine, the glucose oligo- and polysaccharides are broken down to monosaccharides by the pancreatic and intestinal glycosidases. Other polysaccharides cannot be processed by the human intestine and require assistance by intestinal flora if they are to be broken down; the most notable exceptions are sucrose (fructose-glucose) and lactose (galactose-glucose). Glucose is then transported across the apical membrane of the enterocytes by SLC5A1, and later across their basal membrane by SLC2A2. Some of the glucose is directly utilized as an energy source by brain cells, intestinal cells and red blood cells, while the rest reaches the liver, adipose tissue and muscle cells, where it is absorbed and stored as glycogen (under the influence of insulin). Liver cell glycogen can be converted to glucose and returned to the blood when insulin is low or absent; muscle cell glycogen is not returned to the blood because of a lack of enzymes. In fat cells, glucose is used to power reactions that synthesize some fat types and have other purposes. Glycogen is the body's 'glucose energy storage' mechanism because it is much more 'space efficient' and less reactive than glucose itself.

History Because glucose is a basic necessity of many organisms, a correct understanding of its chemical makeup and structure contributed greatly to a general advancement in organic chemistry. This understanding occurred largely as a result of the investigations of Emil Fischer, a German chemist who received the 1902 Nobel Prize in Chemistry as a result of his findings. The synthesis of glucose established the structure of organic material and consequently formed the first definitive validation of Jacobus Henricus van't Hoff's theories of chemical kinetics and the arrangements of chemical bonds in carbon-bearing molecules. Between 1891 and 1894, Fischer established the stereochemical configuration of all the known sugars and correctly predicted the possible isomers, applying van't Hoff's theory of asymmetrical carbon atoms.

Appendix Glucose is a highly complex chemical compound because it can exist in many isomeric forms and each isomer is subject to rotational isomerism.

Chapter- 2

Fructose

D-Fructose

IUPAC name Fructose Other names fruit sugar, levulose, D-fructofuranose, D-fructose, Darabino-hexulose Identifiers CAS number 57-48-7 PubChem 11769129 ChemSpider 388775 UNII 6YSS42VSEV EC-number 200-333-3 ChEMBL CHEMBL604608 SMILES

InChI

Molecular formula Molar mass Melting point

Properties C6H12O6 180.16 g/mol 103 °C

Fructose, or fruit sugar, is a simple monosaccharide found in many foods. It is one of the three important dietary monosaccharides along with glucose and galactose. The organic fructose molecule was first discovered by Augustin-Pierre Dubrunfaut in 1847. Fructose is a white solid that dissolves in water – it is the most water-soluble of all the sugars. Honey, tree fruits, berries, melons, and some root vegetables contain significant amounts of molecular fructose, usually in combination with glucose, stored in the form of sucrose. About 240,000 tonnes of crystalline fructose are produced annually. Fructose is a component of sucrose. Sucrose is a disaccharide derived from the condensation of glucose and fructose. Fructose is derived from the digestion of table sugar (sucrose). D-Fructose

Crystalline fructose and high-fructose corn syrup are often confused as the same product. Crystalline fructose, which is often produced from a fructose-enriched corn syrup, is indeed the monosaccharide. High-fructose corn syrup, however, refers to a family of mixtures of varying amounts of fructose and glucose.

Chemical properties

Figure 1: Relationship between the acyclic and the cyclic (hemiketal) isomers of fructose.

D-

and L-isomers of fructose (open-chain form)

Fructose is a 6-carbon polyhydroxyketone. It is an isomer of glucose, i.e. both have the same molecular formula (C6H12O6) but they differ structurally. Crystalline fructose adopts a cyclic six-membered structure owing to the stability of its hemiketal and internal hydrogen-bonding. This form is formally called D-fructopyranose. In solution, fructose exists as an equilibrium mixture of 70% fructopyranose and about 22% fructofuranose, as well as small amounts of three other forms, including the acylic structure.

Reactions Fructose and fermentation Fructose may be anaerobically fermented by yeast or bacteria. Yeast enzymes convert sugar (glucose, or fructose) to ethanol and carbon dioxide. The carbon dioxide released during fermentation will remain dissolved in water where it will reach equilibrium with carbonic acid unless the fermentation chamber is left open to the air. The dissolved carbon dioxide and carbonic acid produce the carbonation in bottle fermented beverages. Fructose and Maillard reaction Fructose undergoes the Maillard reaction, non-enzymatic browning, with amino acids. Because fructose exists to a greater extent in the open-chain form than does glucose, the initial stages of the Maillard reaction occurs more rapidly than with glucose. Therefore, fructose potentially may contribute to changes in food palatability, as well as other nutritional effects, such as excessive browning, volume and tenderness reduction during cake preparation, and formation of mutagenic compounds. Dehydration

Fructose readily dehydrates to give hydroxymethylfurfural ("HMF"). This process may in future be part of a low-cost, carbon-neutral system to produce replacements for petrol and diesel from plantations.

Physical and functional properties Relative sweetness The primary reason that fructose is used commercially in foods and beverages, besides its low cost, is its high relative sweetness. It is the sweetest of all naturally occurring carbohydrates. Fructose is generally regarded as being 1.73 times as sweet as sucrose. However, it is the 5-ring form of fructose that is sweeter; the 6-ring form tastes about the same as usual table sugar. Warming fructose leads to formation of the 6-ring form.

Figure 2: Relative sweetness of sugars and sweeteners. The sweetness of fructose is perceived earlier than that of sucrose or dextrose, and the taste sensation reaches a peak (higher than sucrose) and diminishes more quickly than sucrose. Fructose can also enhance other flavors in the system. Sweetness synergy Fructose exhibits a sweetness synergy effect when used in combination with other sweeteners. The relative sweetness of fructose blended with sucrose, aspartame, or saccharin is perceived to be greater than the sweetness calculated from individual components.

Fructose solubility and crystallization Fructose has higher solubility than other sugars as well as other sugar alcohols. Fructose is therefore difficult to crystallize from an aqueous solution. Sugar mixes containing fructose, such as candies, are softer than those containing other sugars because of the greater solubility of fructose. Fructose hygroscopicity and humectancy Fructose is quicker to absorb moisture and slower to release it to the environment than sucrose, dextrose, or other nutritive sweeteners. Fructose is an excellent humectant and retains moisture for a long period of time even at low relative humidity (RH). Therefore, fructose can contribute to improved quality, better texture, and longer shelf life to the food products in which it is used. Freezing point Fructose has a greater effect on freezing point depression than disaccharides or oligosaccharides, which may protect the integrity of cell walls of fruit by reducing ice crystal formation. However, this characteristic may be undesirable in soft-serve or hardfrozen dairy desserts. Fructose and starch functionality in food systems Fructose increases starch viscosity more rapidly and achieves a higher final viscosity than sucrose because fructose lowers the temperature required during gelatinizing of starch, causing a greater final viscosity.

Food sources Natural sources of fructose include fruits, vegetables (including sugar cane), and honey. Fructose is often further concentrated from these sources. The highest dietary sources of fructose, besides pure crystalline fructose, are foods containing table sugar (sucrose), high-fructose corn syrup, agave nectar, honey, molasses, maple syrup, and fruit juices, as these have the highest percentages of fructose (including fructose in sucrose) per serving compared to other common foods and ingredients. Fructose exists in foods either as a free monosaccharide, or bound to glucose as sucrose, a disaccharide. Fructose, glucose, and sucrose may all be present in a food; however, different foods will have varying levels of each of these three sugars. The sugar contents of common fruits and vegetables are presented in Table 1. In general, in foods that contain free fructose, the ratio of fructose to glucose is approximately 1:1; that is, foods with fructose usually contain about an equal amount of free glucose. A value that is above 1 indicates a higher proportion of fructose to glucose, and below 1, a lower proportion. Some fruits have larger proportions of fructose to glucose compared to

others. For example, apples and pears contain more than twice as much free fructose as glucose, while for apricots the proportion is less than half as much fructose as glucose. Apple and pear juices are of particular interest to pediatricians because the high concentrations of free fructose in these juices can cause diarrhea in children. The cells (enterocytes) that line children's small intestines have less affinity for fructose absorption than for glucose and sucrose. Unabsorbed fructose creates higher osmolarity in the small intestine, which draws water into the gastrointestinal tract, resulting in osmotic diarrhea. This phenomenon is discussed in greater detail in the Health Effects section. Table 1 also shows the amount of sucrose found in common fruits and vegetables. Sugar cane and sugar beet have a high concentration of sucrose, and are used for commercial preparation of pure sucrose. Extracted cane or beet juice is clarified, removing impurities; and concentrated by removing excess water. The end product is 99.9% pure sucrose. Sucrose-containing sugars include common table white granulated sugar and powdered sugar, as well as brown sugar. Table 1. Sugar content of selected common plant foods (g/100g) Sucrose Fructose/ as a % Total Total Free Free Food Item Sucrose Glucose of Carbohydrate Sugars Fructose Glucose Ratio Total Sugars Fruits Apple 13.8 10.4 5.9 2.4 2.1 2.0 19.9 Apricot 11.1 9.2 0.9 2.4 5.9 0.7 63.5 Banana 22.8 12.2 4.9 5.0 2.4 1.0 20.0 Grapes 18.1 15.5 8.1 7.2 0.2 1.1 1.0 Peach 9.5 8.4 1.5 2.0 4.8 0.9 56.7 Pineapple 13.1 9.9 2.1 1.7 6.0 1.1 60.8 Pear 15.5 9.8 6.2 2.8 0.8 2.1 8.0 Vegetables Beet, Red 9.6 6.8 0.1 0.1 6.5 1.0 96.2 Carrot 9.6 4.7 0.6 0.6 3.6 1.0 70.0 Corn, 19.0 3.2 0.5 0.5 2.1 1.0 64.0 Sweet Red Pepper, 6.0 4.2 2.3 1.9 0.0 1.2 0.0 Sweet Onion, 7.6 5.0 2.0 2.3 0.7 0.9 14.3 Sweet Sweet 20.1 4.2 0.7 1.0 2.5 0.9 60.3 Potato

Yam

27.9

0.5

tr

Sugar Cane

13 - 18 0.2 – 1.0

Sugar Beet

17 - 18 0.1 – 0.5

tr 0.2 – 1.0 0.1 – 0.5

tr

na

tr

11 - 16

1.0

100

16 - 17

1.0

100

Data obtained at. All data with a unit of g (gram) are based on 100 g of a food item. The fructose / glucose ratio is calculated by dividing the sum of free fructose plus half sucrose by the sum of free glucose plus half sucrose. Fructose is also found in the synthetically manufactured sweetener, high-fructose corn syrup (HFCS). Hydrolyzed corn starch is used as the raw material for production of HFCS. Through the enzymatic treatment, glucose molecules are converted into fructose. There are three types of HFCS, each with a different proportion of fructose: HFCS-42, HFCS-55, and HFCS-90. The number for each HFCS corresponds to the percentage of synthesized fructose present in the syrup. HFCS-90 has the highest concentration of fructose, and is typically used to manufacture HFCS-55; HFCS 55 is used as sweetener in soft drinks, while HFCS-42 is used in many processed foods and baked goods.

Commercial sweeteners (carbohydrate content) Sugar Fructose Glucose Sucrose (Fructose-Glucose) Other Sugars Granulated Sugar (50) (50) 100 0 Brown Sugar 1 1 97 1 HFCS-42 42 53 0 5 HFCS-55 55 41 0 4 HFCS-90 90 5 0 5 Honey 50 44 1 5 Maple Syrup 1 4 95 0 Molasses 23 21 53 3 Corn Syrup 0 35 0 0 Data obtained from Kretchmer, N. & Hollenbeck, CB (1991). Sugars and Sweeteners, Boca Raton, FL: CRC Press, Inc. for HFCS, and USDA for fruits and vegetables and the other refined sugars. Cane and beet sugars have been used as the major sweetener in food manufacturing for centuries. However, with the development of HFCS, a significant shift occurred in the type of sweetener consumption. As seen in Figure 3, this change happened in the 1970s. Contrary to the popular belief, however, with the increase of HFCS consumption, the total fructose intake has not dramatically changed. Granulated sugar is 99.9% pure sucrose, which means that it has equal ratio of fructose to glucose. The most commonly used HFCS, 42 and 55, have about equal ratio of fructose to glucose, with minor

differences. HFCS has simply replaced sucrose as a sweetener. Therefore, despite the changes in the sweetener consumption, the ratio of glucose to fructose intake has remained relatively constant.

Figure 3: Adjusted consumption of refined sugar per capita in the U.S.

Fructose digestion and absorption in humans Fructose exists in foods as either a monosaccharide (free fructose) or as a unit of a disaccharide (sucrose). Free fructose is absorbed directly by the intestine; however, when fructose is consumed in the form of sucrose, digestion occurs entirely in the upper small intestine. As sucrose comes into contact with the membrane of the small intestine, the enzyme sucrase catalyzes the cleavage of sucrose to yield one glucose unit and one fructose unit. Fructose is absorbed in the small intestine, then enters the hepatic portal vein and is directed toward the liver.

Figure 4: Hydrolysis of sucrose to glucose and fructose by sucrase. The mechanism of fructose absorption in the small intestine is not completely understood. Some evidence suggests active transport, because fructose uptake has been shown to occur against a concentration gradient. However, the majority of research supports the claim that fructose absorption occurs on the mucosal membrane via facilitated transport involving GLUT5 transport proteins. Since the concentration of fructose is higher in the lumen, fructose is able to flow down a concentration gradient

into the enterocytes, assisted by transport proteins. Fructose may be transported out of the enterocyte across the basolateral membrane by either GLUT2 or GLUT5, although the GLUT2 transporter has a greater capacity for transporting fructose and therefore the majority of fructose is transported out of the enterocyte through GLUT2.

Figure 5: Intestinal sugar transport proteins.

Capacity and rate of absorption The absorption capacity for fructose in monosaccharide form ranges from less than 5 g to 50 g and adapts with changes in dietary fructose intake. Studies show the greatest absorption rate occurs when glucose and fructose are administered in equal quantities. When fructose is ingested as part of the disaccharide sucrose, absorption capacity is much higher because fructose exists in a 1:1 ratio with glucose. It appears that the GLUT5 transfer rate may be saturated at low levels, and absorption is increased through joint absorption with glucose. One proposed mechanism for this phenomenon is a glucosedependent cotransport of fructose. In addition, fructose transfer activity increases with dietary fructose intake. The presence of fructose in the lumen causes increased mRNA transcription of GLUT5, leading to increased transport proteins. High-fructose diets have been shown to increase abundance of transport proteins within 3 days of intake.

Malabsorption

Several studies have measured the intestinal absorption of fructose using hydrogen breath test. These studies indicate that fructose is not completely absorbed in the small intestine. When fructose is not absorbed in the small intestine, it is transported into the large intestine, where it is fermented by the colonic flora. Hydrogen is produced during the fermentation process and dissolves into the blood of the portal vein. This hydrogen is transported to the lungs, where it is exchanged across the lungs and is measurable by the hydrogen breath test. The colonic flora also produces carbon dioxide, short-chain fatty acids, organic acids, and trace gases in the presence of unabsorbed fructose. The presence of gases and organic acids in the large intestine causes gastrointestinal symptoms such as bloating, diarrhea, flatulence, and gastrointestial pain Exercise immediately after consumption can exacerbate these symptoms by decreasing transit time in the small intestine, resulting in a greater amount of fructose being emptied into the large intestine.

Fructose metabolism All three dietary monosaccharides are transported into the liver by the GLUT 2 transporter. Fructose and galactose are phosphorylated in the liver by fructokinase (Km= 0.5 mM) and galactokinase (Km = 0.8 mM). By contrast, glucose tends to pass through the liver (Km of hepatic glucokinase = 10 mM) and can be metabolised anywhere in the body. Uptake of fructose by the liver is not regulated by insulin.

Fructolysis Fructolysis initially produces fructose 1,6-bisphosphate, which is split to produce phosphate derivatives of the trioses dihydroxyacetone and glyceraldehyde. These are then metabolized either in the gluconeogenic pathway for glycogen replenishment and/or complete metabolism in the fructolytic pathway to pyruvate, which after conversion to acetyl-CoA enters the Krebs cycle, and is converted to citrate and subsequently directed toward ’’de novo’’ synthesis of the free fatty acid palmitate.

Metabolism of fructose to DHAP and glyceraldehyde The first step in the metabolism of fructose is the phosphorylation of fructose to fructose 1-phosphate by fructokinase, thus trapping fructose for metabolism in the liver. Fructose 1-phosphate then undergoes hydrolysis by aldolase B to form DHAP and glyceraldehydes; DHAP can either be isomerized to glyceraldehyde 3-phosphate by triosephosphate isomerase or undergo reduction to glycerol 3-phosphate by glycerol 3phosphate dehydrogenase. The glyceraldehyde produced may also be converted to glyceraldehyde 3-phosphate by glyceraldehyde kinase or converted to glycerol 3phosphate by glyceraldehyde 3-phosphate dehydrogenase. The metabolism of fructose at this point yields intermediates in the gluconeogenic and fructolytic pathways leading to glycogen synthesis as well as fatty acid and triglyceride synthesis.

Synthesis of glycogen from DHAP and glyceraldehyde 3 phosphate

The resultant glyceraldehyde formed by aldolase B then undergoes phosphorylation to glyceraldehyde 3-phosphate. Increased concentrations of DHAP and glyceraldehyde 3phosphate in the liver drive the gluconeogenic pathway toward glucose and subsequent glycogen synthesis. It appears that fructose is a better substrate for glycogen synthesis than glucose and that glycogen replenishment takes precedence over triglyceride formation. Once liver glycogen is replenished, the intermediates of fructose metabolism are primarily directed toward triglyceride synthesis.

Figure 6: Metabolic conversion of fructose to glycogen in the liver.

Synthesis of triglyceride from DHAP and glyceraldehyde 3 phosphate Carbons from dietary fructose are found in both the free fatty acid and glycerol moieties of plasma triglycerides. High fructose consumption can lead to excess pyruvate production, causing a buildup of Krebs cycle intermediates. Accumulated citrate can be transported from the mitochondria into the cytosol of hepatocytes, converted to acetyl CoA by citrate lyase and directed toward fatty acid synthesis. Additionally, DHAP can be converted to glycerol 3-phosphate as previously mentioned, providing the glycerol backbone for the triglyceride molecule. Triglycerides are incorporated into very low density lipoproteins (VLDL), which are released from the liver destined toward peripheral tissues for storage in both fat and muscle cells.

Figure 7: Metabolic conversion of fructose to triglyceride in the liver.

Health effects Digestive problems Fructose absorption occurs via the GLUT-5 (fructose only) transporter, and the GLUT2 transporter, for which it competes with glucose and galactose. A deficiency of GLUT 5 may result in excess fructose carried into the lower intestine. There, it can provide nutrients for the existing gut flora, which produce gas. It may also cause water retention in the intestine. These effects may lead to bloating, excessive flatulence, loose stools, and even diarrhea depending on the amounts eaten and other factors. For many people, fructose malabsorption is a major health concern.

Metabolic syndrome Excess fructose consumption has been hypothesized to be a cause of insulin resistance, obesity, elevated LDL cholesterol and triglycerides, leading to metabolic syndrome. Fructose consumption has been shown to be correlated with obesity, especially central

obesity which is thought to be the most dangerous kind of obesity. A study in mice showed that a high fructose intake increases adiposity. Although all simple sugars have nearly identical chemical formulae, each has distinct chemical properties. This can be illustrated with pure fructose. A journal article reports that, "...fructose given alone increased the blood glucose almost as much as a similar amount of glucose (78% of the glucose-alone area)". One study concluded that fructose "produced significantly higher fasting plasma triacylglycerol values than did the glucose diet in men" and "...if plasma triacylglycerols are a risk factor for cardiovascular disease, then diets high in fructose may be undesirable". Bantle et al. "noted the same effects in a study of 14 healthy volunteers who sequentially ate a high-fructose diet and one almost devoid of the sugar." Fructose is a reducing sugar, as are all monosaccharides. The spontaneous chemical reaction of simple sugar molecules to proteins, known as glycation, is thought to be a significant cause of damage in diabetics. Fructose appears to be equivalent to glucose in this regard and so does not seem to be a better answer for diabetes for this reason alone, save for the smaller quantities required to achieve equivalent sweetness in some foods. This may be an important contribution to senescence and many age-related chronic diseases.

Compared to sucrose Studies that have compared high-fructose corn syrup (an ingredient in nearly all soft drinks sold in the US) to sucrose (common table sugar) find that most measured physiological effects are equivalent. For instance, Melanson et al. (2006), studied the effects of HFCS and sucrose sweetened drinks on blood glucose, insulin, leptin, and ghrelin levels. They found no significant differences in any of these parameters. This is not surprising, since sucrose is a disaccharide that digests to 50% fructose and 50% glucose, whereas the high-fructose corn syrup most commonly used on soft drinks is 55% fructose and 45% glucose. The difference between the two lies in the fact that HFCS contains little sucrose, the fructose and glucose being independent moieties. Fructose is often recommended for diabetics because it does not trigger the production of insulin by pancreatic ß cells, probably because ß cells have low levels of GLUT5. Fructose has a very low glycemic index of 19 ± 2, compared with 100 for glucose and 68 ± 5 for sucrose. Fructose is also seventy-three percent sweeter than sucrose at room temperature, so diabetics can use less of it. Studies show that fructose consumed before a meal may even lessen the glycemic response of the meal. Its sweetness changes at higher temperatures, so its effects in recipes are not equivalent to those of sucrose (i.e., table sugar).

Liver disease

"The medical profession thinks fructose is better for diabetics than sugar," says Meira Field, Ph.D., a research chemist at United States Department of Agriculture, "but every cell in the body can metabolize glucose. However, all fructose must be metabolized in the liver. The livers of the rats on the high fructose diet looked like the livers of alcoholics, plugged with fat and cirrhotic." While a few other tissues (e.g., sperm cells and some intestinal cells) do use fructose directly, fructose is almost entirely metabolized in the liver. "When fructose reaches the liver," says Dr. William J. Whelan, a biochemist at the University of Miami School of Medicine, "the liver goes bananas and stops everything else to metabolize the fructose." Eating fructose instead of glucose results in lower circulating insulin and leptin levels, and higher ghrelin levels after the meal. Since leptin and insulin decrease appetite and ghrelin increases appetite, some researchers suspect that eating large amounts of fructose increases the likelihood of weight gain. Excessive fructose consumption is also believed to contribute to the development of nonalcoholic fatty liver disease.

Gout It has been suggested in a recent British Medical Journal study that high consumption of fructose is linked to gout. Cases of gout have risen in recent years, despite commonly being thought of as a Victorian disease, and it is suspected that the fructose found in soft drinks (e.g., carbonated beverages) and other sweetened drinks is the reason for this. In order for the liver to process fructose, it must be phosphorylated by removal of phosphates from adenosine triphosphate (ATP). The ATP gets converted to adenosine monophosphate (AMP), then to inositol monophosphate (IMP), and finally to uric acid, the agent in gout.

Glycaemic index Fructose has the lowest Glycaemic Index (G.I. = 19) of all the natural sugars and may be used in moderation by Diabetics. It is recommended that a maximum intake of 25g - 40g per day is adopted, provided that this is counted towards the daily calorie count. In comparison, ordinary table sugar (Sucrose) has a GI of 65 and Honey has a GI of 55. Fruit Sugar has the advantage for Diabetics that, being nearly twice as sweet as ordinary sugar if not heat-processed, the daily allowance can be stretched by using less for most applications. Many artificial sweeteners are not suitable for home-baking, but, with a little adjustment, many traditional recipes can be prepared using fructose.

Chapter- 3

Glycogen

Schematic 2-D cross-sectional view of glycogen. A core protein of glycogenin is surrounded by branches of glucose units. The entire globular granule may contain approximately 30,000 glucose units.

A view of the atomic structure of a single branched strand of glucose units in a glycogen molecule. Glycogen is the molecule that functions as the secondary long-term energy storage in animal and fungal cells. It is made primarily by the liver and the muscles, but can also be made by glycogenesis within the brain and stomach. Glycogen is the analogue of starch, a less branched glucose polymer in plants, and is commonly referred to as animal starch, having a similar structure to amylopectin. Glycogen is found in the form of granules in the cytosol in many cell types, and plays an important role in the glucose cycle. Glycogen forms an energy reserve that can be quickly mobilized to meet a sudden need for glucose, but one that is less compact than the energy reserves of triglycerides (lipids). In the liver hepatocytes, glycogen can compose up to 8% of the fresh weight (100–120 g in an adult) soon after a meal. Only the glycogen

stored in the liver can be made accessible to other organs. In the muscles, glycogen is found in a low concentration (1% to 2% of the muscle mass). However, the amount of glycogen stored in the body, especially within the red blood cells, liver & muscles, mostly depends on physical training, basal metabolic rate and eating habits such as intermittent fasting. Small amounts of glycogen are found in the kidneys, and even smaller amounts in certain glial cells in the brain and white blood cells. The uterus also stores glycogen during pregnancy to nourish the embryo.

Function and regulation of liver glycogen As a meal containing carbohydrates is eaten and digested, blood glucose levels rise, and the pancreas secretes insulin. Glucose from the portal vein enters liver cells (hepatocytes). Insulin acts on the hepatocytes to stimulate the action of several enzymes, including glycogen synthase. Glucose molecules are added to the chains of glycogen as long as both insulin and glucose remain plentiful. In this postprandial or "fed" state, the liver takes in more glucose from the blood than it releases. After a meal has been digested and glucose levels begin to fall, insulin secretion is reduced, and glycogen synthesis stops. When it is needed for energy, glycogen is broken down and converted again to glucose. Glycogen phosphorylase is the primary enzyme of glycogen breakdown. For the next 8–12 hours, glucose derived from liver glycogen will be the primary source of blood glucose to be used by the rest of the body for fuel. Glucagon is another hormone produced by the pancreas, which in many respects serves as a counter-signal to insulin. When the blood sugar begins to fall below normal, glucagon is secreted in increasing amounts. It stimulates glycogen breakdown into glucose even when insulin levels are abnormally high.

In muscle and other cells Muscle cell glycogen appears to function as an immediate reserve source of available glucose for muscle cells. Other cells that contain small amounts use it locally as well. Muscle cells lack the enzyme glucose-6-phosphatase, which is required to pass glucose into the blood, so the glycogen they store is destined for internal use and is not shared with other cells. (This is in contrast to liver cells, which, on demand, readily do break down their stored glycogen into glucose and send it through the blood stream as fuel for the brain or muscles). Glycogen is also a suitable storage substance due to its insolubility in water, which means it does not affect the osmotistic levels and pressure of a cell.

Glycogen debt and endurance exercise Long-distance athletes such as marathon runners, cross-country skiers, and cyclists often go into glycogen debt, where almost all of the athlete's glycogen stores are depleted after long periods of exertion without enough energy consumption. This phenomenon is referred to as "hitting the wall". In marathon runners, it normally happens around the 20-

mile (32 km) point of a marathon, depending on the size of the runner and the race course. However, it can be delayed by a carbohydrate loading before the task. When experiencing glycogen debt, athletes often experience extreme fatigue to the point that it is difficult to move. A study published in the Journal of Applied Physiology (online May 8, 2008) suggests that, when athletes ingest both carbohydrate and caffeine following exhaustive exercise, their glycogen is replenished more rapidly.

Disorders of glycogen metabolism The most common disease in which glycogen metabolism becomes abnormal is diabetes, in which, because of abnormal amounts of insulin, liver glycogen can be abnormally accumulated or depleted. Restoration of normal glucose metabolism usually normalizes glycogen metabolism as well. In hypoglycemia caused by excessive insulin, liver glycogen levels are high, but the high insulin level prevents the glycogenolysis necessary to maintain normal blood sugar levels. Glucagon is a common treatment for this type of hypoglycemia. Various inborn errors of metabolism are caused by deficiencies of enzymes necessary for glycogen synthesis or breakdown. These are collectively referred to as glycogen storage diseases.

Synthesis

Glycogen Structure Segment Glycogen synthesis is, unlike its breakdown, endergonic. This means that glycogen synthesis requires the input of energy. Energy for glycogen synthesis comes from UTP, which reacts with glucose-1-phosphate, forming UDP-glucose, in reaction catalysed by

UDP-glucose pyrophosphorylase. Glycogen is synthesized from monomers of UDPglucose by the enzyme glycogen synthase, which progressively lengthens the glycogen chain with (α1→4) bonded glucose. As glycogen synthase can lengthen only an existing chain, the protein glycogenin is needed to initiate the synthesis of glycogen. The glycogen-branching enzyme, amylo (α1→4) to (α1→6) transglycosylase, catalyzes the transfer of a terminal fragment of 6-7 glucose residues from a nonreducing end to the C-6 hydroxyl group of a glucose residue deeper into the interior of the glycogen molecule. The branching enzyme can act upon only a branch having at least 11 residues, and the enzyme may transfer to the same glucose chain or adjacent glucose chains.

Breakdown Glycogen is cleaved from the nonreducing ends of the chain by the enzyme glycogen phosphorylase to produce monomers of glucose-1-phosphate, which is then converted to glucose 6-phosphate by phosphoglucosemutase. A special debranching enzyme is needed to remove the alpha(1-6) branches in branched glycogen and reshape the chain into linear polymer. The G6P monomers produced have three possible fates:

• • •

G6P can continue on the glycolysis pathway and be used as fuel. G6P can enter the pentose phosphate pathway via the enzyme Glucose-6phosphate dehydrogenase to produce NADPH and 5-carbon sugars. In the liver and kidney, G6P can be dephosphorylated back to Glucose by the enzyme Glucose 6-phosphatase. This is the final step in the gluconeogenesis pathway.

Chapter- 4

Starch

Starch

CAS number EC-number RTECS number Molecular formula Appearance Density Melting point Solubility in water MSDS EU Index Autoignition temperature

Identifiers 9005-25-8 232-679-6 GM5090000 Properties (C6H10O5)n white powder 1.5 g/cm3 decomp. none Hazards ICSC 1553 not listed 410 °C

Structure of the amylose molecule.

Structure of the amylopectin molecule.

Granules of wheat starch, stained with iodine, photographed through a light microscope. Starch or amylum is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. This polysaccharide is produced by all green plants as an energy store. It is the most important carbohydrate in the human diet and is contained in such staple foods as potatoes, wheat, maize (corn), rice, and cassava.

Starch

Pure starch is a white, tasteless and odorless powder that is insoluble in cold water or alcohol. It consists of two types of molecules: the linear and helical amylose and the branched amylopectin. Depending on the plant, starch generally contains 20 to 25% amylose and 75 to 80% amylopectin. Glycogen, the glucose store of animals, is a more branched version of amylopectin. Starch is processed to produce many of the sugars in processed foods. When dissolved in warm water, it can be used as a thickening, stiffening or gluing agent, giving wheatpaste.

Name The word "starch" is derived from Middle English sterchen, meaning to stiffen. "Amylum" is Latin for starch, from the Greek "amulon" which means "not ground at a mill". The root amyl is used in biochemistry for several compounds related to starch.

History Starch grains from the rhizomes of Typha (cattails, bullrushes) have been identified from grinding stones in Europe dating back to 30,000 years ago.

Wheat starch paste was used by Egyptians to stiffen cloth and during weaving linen and possibly to glue papyrus. Romans used it also in cosmetic creams, to powder the hair and to thicken sauces. Persians and Indians used it to make dishes similar to gothumai wheat halva. In China, with the invention of paper, rice starch was used as a surface treatment of the paper.

Energy store of plants In photosynthesis, plants use light energy to produce glucose from carbon dioxide. The glucose is stored mainly in the form of starch granules, in plastids such as chloroplasts and especially amyloplasts. Toward the end of the growing season, starch accumulates in twigs of trees near the buds. Fruit, seeds, rhizomes, and tubers store starch to prepare for the next growing season. Glucose is soluble in water, hydrophilic, binds much water and then takes up much space; glucose in the form of starch, on the other hand, is not soluble and can be stored much more compactly. Glucose molecules are bound in starch by the easily hydrolyzed alpha bonds. The same type of bond can also be seen in the animal reserve polysaccharide glycogen. This is in contrast to many structural polysaccharides such as chitin, cellulose and peptidoglycan, which are bound by beta-bonds and are much more resistant to hydrolysis.

Biosynthesis Plants produce starch by first converting glucose 1-phosphate to ADP-glucose using the enzyme glucose-1-phosphate adenylyltransferase. This step requires energy in the form of ATP. The enzyme starch synthase then adds the ADP-glucose via a 1,4-alpha glycosidic bond to a growing chain of glucose residues, liberating ADP and creating amylose. Starch branching enzyme introduces 1,6-alpha glycosidic bonds between these chains, creating the branched amylopectin. The starch debranching enzyme isoamylase removes some of these branches. Several isoforms of these enzymes exist, leading to a highly complex synthesis process. While amylose was traditionally thought to be completely unbranched, it is now known that some of its molecules contain a few branch points. Glycogen and amylopectin have the same structure, but the former has about one branch point per ten 1,4-alpha bonds, compared to about one branch point per thirty 1,4-alpha bonds in amylopectin. Another difference is that glycogen is synthesised from UDPglucose while starch is synthesised from ADP-glucose.

Properties Structure

Starch molecules arrange themselves in the plant in semi-crystalline granules. Each plant species has a unique starch granular size: rice starch is relatively small (about 2μm) while potato starches have larger granules (up to 100μm). Although in absolute mass only about one quarter of the starch granules in plants consist of amylose, there are about 150 times more amylose molecules than amylopectin molecules. Amylose is a much smaller molecule than amylopectin. Starch becomes soluble in water when heated. The granules swell and burst, the semicrystalline structure is lost and the smaller amylose molecules start leaching out of the granule, forming a network that holds water and increasing the mixture's viscosity. This process is called starch gelatinization. During cooking the starch becomes a paste and increases further in viscosity. During cooling or prolonged storage of the paste, the semicrystalline structure partially recovers and the starch paste thickens, expelling water. This is mainly caused by the retrogradation of the amylose. This process is responsible for the hardening of bread or staling, and for the water layer on top of a starch gel (syneresis). Some cultivated plant varieties have pure amylopectin starch without amylose, known as waxy starches. The most used is waxy maize, others are glutinous rice and waxy potato starch. Waxy starches have less retrogradation, resulting in a more stable paste. High amylose starch, amylomaize, is cultivated for the use of its gel strength.

Hydrolysis The enzymes that break down or hydrolyze starch into the constituent sugars are known as amylases. Alpha-amylases are found in plants and in animals. Human saliva is rich in amylase, and the pancreas also secretes the enzyme. Individuals from populations with a high-starch diet tend to have more amylase genes than those with low-starch diets ; chimpanzees have very few amylase genes . It is possible that turning to a high-starch diet was a significant event in human evolution. Beta-amylase cuts starch into maltose units. This process is important in the digestion of starch and is also used in brewing, where the amylase from the skin of the seed grains is responsible for converting starch to maltose (Malting, Mashing).

Dextrinization If starch is subjected to dry heat, it breaks down to form pyrodextrins, in a process known as dextrinization. Pyrodextrins are brown in color. This process is partially responsible for the browning of toasted bread.

Chemical tests Iodine solution is used to test for starch; a dark blue color indicates the presence of starch. The details of this reaction are not yet fully known, but it is thought that the iodine

(I3− and I5− ions) fit inside the coils of amylose, the charge transfers between the iodine and the starch, and the energy level spacings in the resulting complex correspond to the absorption spectrum in the visible light region. The strength of the resulting blue color depends on the amount of amylose present. Waxy starches with little or no amylose present will color red.

Starch, 800x magnified, under polarized light. Starch indicator solution consisting of water, starch and iodine is often used in redox titrations: in the presence of an oxidizing agent the solution turns blue, in the presence of reducing agent the blue color disappears because triiodide (I3−) ions break up into three iodide ions, disassembling the starch-iodine complex. A 0.3% w/w solution is the standard concentration for a starch indicator. It is made by adding 3 grams of soluble starch to 1 litre of heated water; the solution is cooled before use (starch-iodine complex becomes unstable at temperatures above 35 °C). Microscopy of starch granules - Each species of plant has a unique shape of starch granules in granular size, shape and crystallisation pattern. Under the microscope, starch grains stained with iodine illuminated from behind with polarized light show a distinctive Maltese cross effect (also known as extinction cross and birefringence).

Starch as food

Starch is the most important carbohydrate in the human diet and is contained in many staple foods. The major sources of starch intake worldwide are rice, wheat, maize (corn), potatoes and cassava. Widely used prepared foods containing starch are bread, pancakes, cereals, noodles, pasta, porridge and tortilla. Depending on the local climate other starch sources are used for food, such as acorn, arrowroot, arracacha, banana, barley, breadfruit, buckwheat, canna, colacasia, katakuri, kudzu, malanga, millet, oat, oca, polynesian arrowroot, sago, sorghum, sweet potato, rye, taro, water chestnut and yams. Chestnuts and edible beans, such as favas, lentils, mung bean and peas, are also rich in starch. Digestive enzymes have problems digesting crystalline structures. Raw starch will digest poorly in the duodenum and small intestine, while bacterial degradation will take place mainly in the colon. Resistant starch is starch that escapes digestion in the small intestine of healthy individuals. In order to increase the digestibility, starch is cooked. Hence, before humans started using fire, eating grains was not a very useful way to get energy.

Starch industry The starch industry extracts and refines starches from seeds, roots and tubers, by wet grinding, washing, sieving and drying. Today, the main commercial refined starches are cornstarch, tapioca, wheat and potato starch. To a lesser extent, sources include rice, sweet potato, sago and mung bean. Historically, Florida arrowroot was also commercialized. Starch is still extracted from more than 50 types of plants. Untreated starch requires heat to thicken or gelatinize. When a starch is pre-cooked, it can then be used to thicken instantly in cold water. This is referred to as a pregelatinized starch. Starch sugars Starch can be hydrolyzed into simpler carbohydrates by acids, various enzymes, or a combination of the two. The resulting fragments are known as dextrins. The extent of conversion is typically quantified by dextrose equivalent (DE), which is roughly the fraction of the glycosidic bonds in starch that have been broken. These starch sugars are by far the most common starch based food ingredient and are used as sweetener in many drinks and foods. They include: • • •

Maltodextrin, a lightly hydrolyzed (DE 10–20) starch product used as a blandtasting filler and thickener. Various glucose syrup / corn syrups (DE 30–70), viscous solutions used as sweeteners and thickeners in many kinds of processed foods. Dextrose (DE 100), commercial glucose, prepared by the complete hydrolysis of starch.

•

•

High fructose syrup, made by treating dextrose solutions with the enzyme glucose isomerase, until a substantial fraction of the glucose has been converted to fructose. In the United States, high fructose corn syrup is the principal sweetener used in sweetened beverages because fructose has better handling characteristics, such as microbiological stability, and more consistent sweetness/flavor. High fructose corn syrup is typically more sweet than regular sucrose because more HFCS-55 is made (which is sweeter) than HFC-42 (on par with sugar). Sugar alcohols, such as maltitol, erythritol, sorbitol, mannitol and hydrogenated starch hydrolysate, are sweeteners made by reducing sugars.

Modified starches A modified food starch is a starch that has been chemically modified to allow the starch to function properly under conditions frequently encountered during processing or storage, such as high heat, high shear, low pH, freeze/thaw and cooling. The modified starches are E coded according to the International Numbering System for Food Additives (INS): • • • • • • • • • • • • • • • •

1401 Acid-treated starch 1402 Alkaline-treated starch 1403 Bleached starch 1404 Oxidized starch 1405 Starches, enzyme-treated 1410 Monostarch phosphate 1412 Distarch phosphate 1413 Phosphated distarch phosphate 1414 Acetylated distarch phosphate 1420 Starch acetate 1422 Acetylated distarch adipate 1440 Hydroxypropyl starch 1442 Hydroxypropyl distarch phosphate 1443 Hydroxypropyl distarch glycerol 1450 Starch sodium octenyl succinate 1451 Acetylated oxidized starch

INS 1401, 1402, 1403 and 1405 are in the EU food ingredients without an E-number. Typical modified starches for technical applications are cationic starches, hydroxyethyl starch and carboxymethylated starches. Use as food additive As an additive for food processing, food starches are typically used as thickeners and stabilizers in foods such as puddings, custards, soups, sauces, gravies, pie fillings, and salad dressings, and to make noodles and pastas.

Gummed sweets such as jelly beans and wine gums are not manufactured using a mold in the conventional sense. A tray is filled with native starch and leveled. A positive mold is then pressed into the starch leaving an impression of 1000 or so jelly beans. The jelly mix is then poured into the impressions and put into a stove to set. This method greatly reduces the number of molds that must be manufactured. In the pharmaceutical industry, starch is also used as an excipient, as tablet disintegrant or as binder.

Industrial applications

Starch adhesive. Papermaking is the largest non-food application for starches globally, consuming millions of metric tons annually. In a typical sheet of copy paper for instance, the starch content may be as high as 8%. Both chemically modified and unmodified starches are used in papermaking. In the wet part of the papermaking process, generally called the “wet-end”, the starches used are cationic and have a positive charge bound to the starch polymer. These starch derivatives associate with the anionic or negatively charged paper fibers / cellulose and inorganic fillers. Cationic starches together with other retention and internal sizing agent help to give the necessary strength properties to the paper web to be

formed in the papermaking process (wet strength), and to provide strength to the final paper sheet (dry strength). In the dry end of the papermaking process the paper web is rewetted with a starch based solution. The process is called surface sizing. Starches used have been chemically, or enzymatically depolymerized at the paper mill or by the starch industry (oxidized starch). The size - starch solutions are applied to the paper web by means of various mechanical presses (size press). Together with surface sizing agent the surface starches impart additional strength to the paper web and additionally provide water hold out or “size” for superior printing properties. Starch is also used in paper coating as one of the binders for the coating formulation a mixture of pigments, binders and thickeners. Coated paper has improved smoothness, hardness, whiteness and gloss and thus improves printing characteristics. Corrugated board adhesives are the next largest application of non-food starches globally. Starch glues are mostly based on unmodified native starches, plus some additive such as borax and caustic soda. Part of the starch is gelatinized to carry the slurry of uncooked starches and prevent sedimentation. This opaque glue is called a SteinHall adhesive. The glue is applied on tips of the fluting. The fluted paper is pressed to paper called liner. This is then dried under high heat, which causes the rest of the uncooked starch in glue to swell/gelatinize. This gelatinizing makes the glue a fast and strong adhesive for corrugated board production. Another large non-food starch application is in the construction industry, where starch is used in the gypsum wall board manufacturing process. Chemically modified or unmodified starches are added to the stucco containing primarily gypsum. Top and bottom heavyweight sheets of paper are applied to the formulation, and the process is allowed to heat and cure to form the eventual rigid wall board. The starches act as a glue for the cured gypsum rock with the paper covering, and also provide rigidity to the board. Starch is used in the manufacture of various adhesives or glues for book-binding, wallpaper adhesives, paper sack production, tube winding, gummed paper, envelop adhesives, school glues and bottle labeling. Starch derivatives, such as yellow dextrins, can be modified by addition of some chemicals to form a hard glue for paper work; some of those forms use borax or soda ash, which are mixed with the starch solution at 50-70 °C to create a very good adhesive. Sodium silicate can be added to reinforce these formulae. Clothing starch or laundry starch is a liquid that is prepared by mixing a vegetable starch in water (earlier preparations also had to be boiled), and is used in the laundering of clothes. Starch was widely used in Europe in the 16th and 17th centuries to stiffen the wide collars and ruffs of fine linen which surrounded the necks of the well-to-do. During the 19th century and early 20th century, it was stylish to stiffen the collars and sleeves of men's shirts and the ruffles of girls' petticoats by applying starch to them as the clean clothes were being ironed. Aside from the smooth, crisp edges it gave to clothing, it

served practical purposes as well. Dirt and sweat from a person's neck and wrists would stick to the starch rather than to the fibers of the clothing, and would easily wash away along with the starch. After each laundering, the starch would be reapplied. Today, the product is sold in aerosol cans for home use. Starch is also used to make some packing peanuts, and some drop ceiling tiles. Textile chemicals from starch are used to reduce breaking of yarns during weaving; the warp yarns are sized, especially for cotton. Starch is also used as textile printing thickener. In the printing industry, food grade starch is used in the manufacture of anti-set-off spray powder used to separate printed sheets of paper to avoid wet ink being set off. Starch is used to produce various bioplastics, synthetic polymers that are biodegradable. An example is polylactic acid. For body powder, powdered corn starch is used as a substitute for talcum powder, and similarly in other health and beauty products. In oil exploration, starch is used to adjust the viscosity of drilling fluid, which is used to lubricate the drill head and suspend the grinding residue in petroleum extraction. Glucose from starch can be further fermented to biofuel ethanol. Hydrogen production can use starch as the raw material, using enzymes.

Chapter- 5

Pyruvic Acid

Pyruvic acid

IUPAC name 2-oxopropanoic acid Other names α-ketopropionic acid; acetylformic acid; pyroracemic acid; Pyr Identifiers CAS number 127-17-3 PubChem 1060 ChemSpider 1031 UNII 8558G7RUTR Properties Molecular formula C3H4O3 Molar mass 88.06 g/mol Density 1.250 g/cm³ Melting point 11.8 °C, 285 K, 53 °F Boiling point 165 °C, 438 K, 329 °F Acidity (pKa) 2.50 Related compounds pyruvate ion Other anions

Related keto-acids, carboxylic acids Related compounds

acetic acid glyoxylic acid oxalic acid propionic acid acetoacetic acid propionaldehyde

glyceraldehyde methylglyoxal sodium pyruvate

Pyruvic acid (CH3COCOOH) is an organic acid, a ketone, as well the simplest of the alpha-keto acids. The carboxylate (COOH) ion (anion) of pyruvic acid, CH3COCOO-, is known as pyruvate, and is a key intersection in several metabolic pathways. It can be made from glucose through glycolysis, supplies energy to living cells in the citric acid cycle (also known as the Krebs cycle), and can also be converted to carbohydrates via gluconeogenesis, to fatty acids or energy through acetyl-CoA, to the amino acid alanine and to ethanol.

Chemistry In 1834, Théophile-Jules Pelouze distilled both tartaric acid (L-tartaric acid) and racemic acid (a mix of D- and L-tartaric acid) and isolated pyrotartaric acid (methyl succinic acid ) and another acid which Jöns Jacob Berzelius characterized the following year and named pyruvic acid. Pyruvic acid is a colorless liquid with a smell similar to that of acetic acid and is miscible with water. In the laboratory, pyruvic acid may be prepared by heating a mixture of tartaric acid and potassium hydrogen sulfate, by the oxidation of propylene glycol by a strong oxidizer (eg. potassium permanganate or bleach), or by the hydrolysis of acetyl cyanide, formed by reaction of acetyl chloride with potassium cyanide: CH3COCl + KCN → CH3COCN + KCl CH3COCN → CH3COCOOH

Biochemistry Pyruvate is an important chemical compound in biochemistry. It is the output of the anaerobic metabolism of glucose known as glycolysis. One molecule of glucose breaks down into two molecules of pyruvate, which are then used to provide further energy, in one of two ways. Pyruvate is converted into acetyl-coenzyme A, which is the main input for a series of reactions known as the Krebs cycle. Pyruvate is also converted to oxaloacetate by an anaplerotic reaction which replenishes Krebs cycle intermediates; alternatively, the oxaloacetate is used for gluconeogenesis. These reactions are named after Hans Adolf Krebs, the biochemist awarded the 1953 Nobel Prize for physiology, jointly with Fritz Lipmann, for research into metabolic processes. The cycle is also called the citric acid cycle, because citric acid is one of the intermediate compounds formed during the reactions. If insufficient oxygen is available, the acid is broken down anaerobically, creating lactate in animals and ethanol in plants and microorganisms. Pyruvate from glycolysis is converted by anaerobic respiration to lactate using the enzyme lactate dehydrogenase and the coenzyme NADH in lactate fermentation, or to acetaldehyde and then to ethanol in alcoholic fermentation.

Pyruvate is a key intersection in the network of metabolic pathways. Pyruvate can be converted into carbohydrates via gluconeogenesis, to fatty acids or energy through acetylCoA, to the amino acid alanine and to ethanol. Therefore it unites several key metabolic processes. The pyruvic acid derivative bromopyruvic acid is being studied for potential cancer treatment applications by researchers at Johns Hopkins University in ways that would support the Warburg hypothesis on the cause(s) of cancer.

Pyruvate production by glycolysis In glycolysis, phosphoenolpyruvate (PEP) is converted to pyruvate by pyruvate kinase. This reaction is strongly exergonic and irreversible; in gluconeogenesis it takes two enzymes, pyruvate carboxylase and PEP carboxykinase, to catalyze the reverse transformation of pyruvate to PEP. phosphoenolpyruvate pyruvate kinase pyruvate ADP

ATP

ADP

ATP

pyruvate kinase Compound C00074 at KEGG Pathway Database. Enzyme 2.7.1.40 at KEGG Pathway Database. Compound C00022 at KEGG Pathway Database.

Pyruvate decarboxylation to acetyl CoA Pyruvate decarboxylation by the pyruvate dehydrogenase complex produces acetyl-CoA. pyruvate pyruvate dehydrogenase complex acetyl-CoA CoA + NAD+ CO2 + NADH + H+

Pyruvate carboxylation to oxaloacetate Carboxylation by the pyruvate carboxylase produces oxaloacetate. pyruvate pyruvate carboxylase oxaloacetate ATP + CO2 ADP + Pi

Transamination by the alanine aminotransferase pyruvate

alanine transaminase

alanine

glutamate α-ketoglutarate

glutamate α-ketoglutarate

Reduction to lactic acid Reduction by the lactate dehydrogenase produces lactic acid. pyruvate lactate dehydrogenase NADH

NAD+

lactic acid

NADH

NAD+

Interactive pathway map

Citric acid cycle

Origin of life Current evolutionary theory on the origin of life posits that the first organisms were anaerobic because the atmosphere of prebiotic Earth was, in theory, almost barren of diatomic oxygen. As such, requisite biochemical materials must have preceded life. In vitro, iron sulfide at sufficient pressure and temperature catalyzes the formation of pyruvate. Thus, argues Günter Wächtershäuser, the mixing of iron-rich crust with hydrothermal vent fluid is suspected of providing the fertile basis for the formation of life.

Chapter- 6

Urea

Urea

IUPAC name Urea Other names Carbamide, carbonyl diamide, carbonyldiamine, diaminomethanal, diaminomethanone Identifiers CAS number 57-13-6 PubChem 1176 ChemSpider 1143 UNII 8W8T17847W RTECS number YR6250000 Properties Molecular formula CH4N2O Molar mass 60.06 g mol−1 Appearance White solid Density 1.32 g/cm3 Melting point 133–135 °C 51,8 g/100 ml (20 °C) Solubility in water 71,7 g/100 ml (60 °C) 95,0 g/100 ml (120 °C) Basicity (pKb) pKBH+ = 0.18 Structure Dipole moment 4.56 D Hazards

JT Baker Not listed Non-flammable Related compounds Thiourea Related ureas Hydroxycarbamide Carbamide peroxide Related compounds Urea phosphate MSDS EU Index Flash point

Urea or carbamide is an organic compound with the chemical formula (NH2)2CO. The molecule has two amine (-NH2) groups joined by a carbonyl (C=O) functional group. Urea serves an important role in the metabolism of nitrogen-containing compounds by animals and is the main nitrogen-containing substance in the urine of mammals. It is solid, colourless, and odorless (although the ammonia which it gives off in the presence of water, including water vapor in the air, has a strong odor). It is highly soluble in water and non-toxic. Dissolved in water it is neither acidic nor alkaline. The body uses it in many processes, most notably nitrogen excretion. Urea is widely used in fertilizers as a convenient source of nitrogen. Urea is also an important raw material for the chemical industry. The synthesis of this organic compound by Friedrich Wöhler in 1828 from an inorganic precursor was an important milestone in the development of organic chemistry, as it showed for the first time that a molecule found in living organisms could be synthesized in the lab without biological starting materials. The terms urea and carbamide are also used for a class of chemical compounds sharing the same functional group RR'N-CO-NRR', namely a carbonyl group attached to two organic amine residues. Examples include carbamide peroxide, allantoin, and hydantoin. Ureas are closely related to biurets and related in structure to amides, carbamates, carbodiimides, and thiocarbamides.

History Urea was first discovered in urine in 1773 by the French chemist Hilaire Rouelle. In 1828, the German chemist Friedrich Wöhler obtained urea by treating silver isocyanate with ammonium chloride. AgNCO + NH4Cl → (NH2)2CO + AgCl This was the first time an organic compound was artificially synthesized from inorganic starting materials, without the involvement of living organisms. The results of this experiment implicitly discredited vitalism: the theory that the chemicals of living organisms are fundamentally different from inanimate matter. This insight was important for the development of organic chemistry. His discovery prompted Wöhler to write triumphantly to Berzelius: "I must tell you that I can make urea without the use of kidneys, either man or dog. Ammonium cyanate is urea." For this discovery, Wöhler is considered by many the father of organic chemistry.

Physiology Urea is synthesized in the body of many organisms as part of the urea cycle, either from the oxidation of amino acids or from ammonia. In this cycle, amino groups donated by ammonia and L-aspartate are converted to urea, while L-ornithine, citrulline, Largininosuccinate, and L-arginine act as intermediates. Urea production occurs in the liver and is regulated by N-acetylglutamate. Urea is found dissolved in blood (in the reference range of 2.5 to 7.5 mmol/liter) and is excreted by the kidney as a component of urine. In addition, a small amount of urea is excreted (along with sodium chloride and water) in sweat. Amino acids from ingested food which are not used for the synthesis of proteins and other biological substances are oxidized by the body, yielding urea and carbon dioxide, as an alternative source of energy. The oxidation pathway starts with the removal of the amino group by a transaminase, the amino group is then fed into the urea cycle. Ammonia (NH3) is another common byproduct of the metabolism of nitrogenous compounds. Ammonia is smaller, more volatile and more mobile than urea. If allowed to accumulate, ammonia would raise the pH in cells to toxic levels. Therefore many organisms convert ammonia to urea, even though this synthesis has a net energy cost. Being practically neutral and highly soluble in water, urea is a safe vehicle for the body to transport and excrete excess nitrogen. In water, the amine groups undergo slow displacement by water molecules, producing ammonia and carbonate anion. For this reason, old, stale urine has a stronger odor than fresh urine.

In humans The handling of urea by the kidneys is a vital part of human metabolism. Besides its role as carrier of waste nitrogen, urea also plays a role in the countercurrent exchange system of the nephrons, that allows for reabsorption of water and critical ions from the excreted urine. Urea is reabsorbed in the inner medullary collecting ducts of the nephrons, thus raising the osmolarity in the medullary interstitium surrounding the thin ascending limb of the loop of Henle, which in turn causes water to be reabsorbed. By action of the urea transporter 2, some of this reabsorbed urea will eventually flow back into the thin ascending limb of the tubule, through the collecting ducts, and into the excreted urine. This mechanism, which is controlled by the antidiuretic hormone, allows the body to create hyperosmotic urine, that has a higher concentration of dissolved substances than the blood plasma. This mechanism is important to prevent the loss of water, to maintain blood pressure, and to maintain a suitable concentration of sodium ions in the blood plasmas. The equivalent nitrogen content (in gram) of urea (in mmol) can be estimated by the conversion factor 0.028 g/mmol. Furthermore, 1 gram of nitrogen is roughly equivalent

to 6 gram of protein, and 1 gram of protein is roughly equivalent to 4 gram of muscle tissue. Subsequently, in situations such as muscle wasting, 1 mmol of excessive urea in the urine (as measured by urine volume in litres multiplied by urea concentration in mmol/l) roughly corresponds to a muscle loss of 0,67 gram.

In other species In aquatic organisms the most common form of nitrogen waste is ammonia, while landdwelling organisms convert the toxic ammonia to either urea or uric acid. Urea is found in the urine of mammals and amphibians, as well as some fish. Birds and saurian reptiles have a different form of nitrogen metabolism, that requires less water and leads to nitrogen being excreted in the form of uric acid. It is noteworthy that tadpoles excrete ammonia but shift to urea production during metamorphosis. Despite the generalization above, the urea pathway has been documented not only in mammals and amphibians but in many other organisms as well, including birds, invertebrates, insects, plants, yeast, fungi, and even microorganisms.

Uses Agriculture More than 90% of world production of urea is destined for use as a nitrogen-release fertilizer. Urea has the highest nitrogen content of all solid nitrogenous fertilizers in common use. Therefore, it has the lowest transportation costs per unit of nitrogen nutrient. Many soil bacteria possess the enzyme, urease, which catalyzes the conversion of the urea molecule to two ammonia molecules and one carbon dioxide molecule, thus urea fertilizers are very rapidly transformed to the ammonium form in soils. Among soil bacteria known to carry urease, some ammonia-oxidizing bacteria (AOB), such as species of Nitrosomonas are also able to assimilate the carbon dioxide released by the reaction to make biomass via the Calvin Cycle, and harvest energy by oxidizing ammonia (the other product of urease) to nitrite, a process termed nitrification. Nitrite-oxidizing bacteria, especially, Nitrobacter, oxidize nitrite to nitrate, which is extremely mobile in soils and is a major cause of water pollution from agriculture. Ammonia and nitrate are readily absorbed by plants, and are the dominant sources of nitrogen for plant growth. Urea is also used in many multi-component solid fertilizer formulations. Urea is highly soluble in water and is, therefore, also very suitable for use in fertilizer solutions (in combination with ammonium nitrate: UAN), e.g., in 'foliar feed' fertilizers. For fertilizer use, granules are preferred over prills because of their narrower particle size distribution which is an advantage for mechanical application. The most common impurity of synthetic urea is biuret, which impairs plant growth. Urea is usually spread at rates of between 40 and 300 kg/ha but rates vary. Smaller applications incur lower losses due to leaching. During summer, urea is often spread just

before, or during rain to minimize losses from volatilization (process wherein nitrogen is lost to the atmosphere as ammonia gas). Urea is not compatible with other fertilizers. Because of the high nitrogen concentration in urea, it is very important to achieve an even spread. The application equipment must be correctly calibrated and properly used. Drilling must not occur on contact with or close to seed, due to the risk of germination damage. Urea dissolves in water for application as a spray or through irrigation systems. In grain and cotton crops, urea is often applied at the time of the last cultivation before planting. In high rainfall areas and on sandy soils (where nitrogen can be lost through leaching) and where good in-season rainfall is expected, urea can be side- or top-dressed during the growing season. Top-dressing is also popular on pasture and forage crops. In cultivating sugarcane, urea is side-dressed after planting, and applied to each ratoon crop. In irrigated crops, urea can be applied dry to the soil, or dissolved and applied through the irrigation water. Urea will dissolve in its own weight in water, but it becomes increasingly difficult to dissolve as the concentration increases. Dissolving urea in water is endothermic, causing the temperature of the solution to fall when urea dissolves. As a practical guide, when preparing urea solutions for fertigation (injection into irrigation lines), dissolve no more than 30 kg urea per 100 L water. In foliar sprays, urea concentrations of 0.5% – 2.0% are often used in horticultural crops. Low-biuret grades of urea are often indicated. Urea absorbs moisture from the atmosphere and therefore is typically stored either in closed/sealed bags on pallets, or, if stored in bulk, under cover with a tarpaulin. As with most solid fertilizers, storage in a cool, dry, well-ventilated area is recommended.

Chemical industry Urea is a raw material for the manufacture of many important chemical compounds, such as • • • •

Various plastics, especially the urea-formaldehyde resins. Various adhesives, such as urea-formaldehyde or the urea-melamineformaldehyde used in marine plywood. Potassium cyanate, another industrial feedstock. Urea nitrate, an explosive.

Automobile systems Urea is used in SNCR and SCR reactions to reduce the NOx pollutants in exhaust gases from combustion from diesel, dual fuel, and lean-burn natural gas engines. The BlueTec system, for example, injects water-based urea solution into the exhaust system. The

ammonia produced by the hydrolysis of the urea reacts with the nitrogen oxide emissions and is converted into nitrogen and water within the catalytic converter.

Other commercial uses • • • • • • • • • • • • • • • •

A stabilizer in nitrocellulose explosives A component of animal feed, providing a relatively cheap source of nitrogen to promote growth A non-corroding alternative to rock salt for road de-icing, and the resurfacing of snowboarding halfpipes and terrain parks A flavor-enhancing additive for cigarettes A main ingredient in hair removers such as Nair and Veet A browning agent in factory-produced pretzels An ingredient in some skin cream, moisturizers, hair conditioners A reactant in some ready-to-use cold compresses for first-aid use, due to the endothermic reaction it creates when mixed with water A cloud seeding agent, along with other salts A flame-proofing agent, commonly used in dry chemical fire extinguisher charges such as the urea-potassium bicarbonate mixture An ingredient in many tooth whitening products An ingredient in dish soap Along with ammonium phosphate, as a yeast nutrient, for fermentation of sugars into ethanol A nutrient used by plankton in ocean nourishment experiments for geoengineering purposes As an additive to extend the working temperature and open time of hide glue As a solubility-enhancing and moisture-retaining additive to dye baths for textile dyeing or printing

Laboratory uses Urea in concentrations up to 10 M is a powerful protein denaturant as it disrupts the noncovalent bonds in the proteins. This property can be exploited to increase the solubility of some proteins. A mixture of urea and choline chloride is used as a deep eutectic solvent, a type of ionic liquid. Urea can in principle serve as a hydrogen source for subsequent power generation in fuel cells. Urea present in urine/wastewater can be used directly (though bacteria normally quickly degrade urea.) Producing hydrogen by electrolysis of urea solution occurs at a lower voltage (0.37v) and thus consumes less energy than the electrolysis of water (1.2v).

Medical use Urea is used in topical dermatological products to promote rehydration of the skin. If covered by an occlusive dressing, 40% urea preparations may also be used for nonsurgical debridement of nails. This drug is also used as an earwax removal aid.

Certain types of instant cold packs (or ice packs) contain water and separated urea crystals. Rupturing the internal water bag starts an endothermic reaction and allows the pack to be used to reduce swelling. Like saline, urea injection is used to perform abortions. Urea is the main component of an alternative medicinal treatment referred to as urine therapy. The blood urea nitrogen (BUN) test is a measure of the amount of nitrogen in the blood that comes from urea. It is used as a marker of renal function. Urea labeled with carbon-14 or carbon-13 is used in the urea breath test, which is used to detect the presence of the bacteria Helicobacter pylori (H. pylori) in the stomach and duodenum of humans, associated with peptic ulcers. The test detects the characteristic enzyme urease, produced by H. pylori, by a reaction that produces ammonia from urea. This increases the pH (reduces acidity) of the stomach environment around the bacteria. Similar bacteria species to H. pylori can be identified by the same test in animals such as apes, dogs, and cats (including big cats).

Analysis Urea is readily quantified by a number of different methods, such as the diacetyl monoxime colorimetric method, and the Berthelot reaction (after initial conversion of urea to ammonia via urease). These methods are amenable to high throughput instrumentation, such as automated flow injection analyzers and 96-well micro-plate spectrophotometers.

Terrorism Ahmed Ressam, the al-Qaeda Millennium Bomber, used urea as one of the components in the explosives that he prepared to bomb Los Angeles International Airport on New Year's Eve 1999/2000; the explosives could have produced a blast 40 times greater than that of a devastating car bomb.

Synthetic production Urea is produced on a scale of some 100,000,000 tons per year worldwide.

Industrial methods For use in industry, urea is produced from synthetic ammonia and carbon dioxide. Large quantities of carbon dioxide are produced during the manufacture of ammonia from coal or from hydrocarbons such as natural gas and petroleum-derived raw materials. Such point sources of CO2 facilitate direct synthesis of urea.

The basic process, developed in 1922, is also called the Bosch-Meiser urea process after its discoverers. The various urea processes are characterized by the conditions under which urea formation takes place and the way in which unconverted reactants are further processed. The process consists of two main equilibrium reactions, with incomplete conversion of the reactants. The first is an exothermic reaction of liquid ammonia with dry ice to form ammonium carbamate (H2N-COONH4): 2 NH3 + CO2 ↔ H2N-COONH4 () The second is an endothermic decomposition of ammonium carbamate into urea and water: H2N-COONH4 ↔ (NH2)2CO + H2O Both reactions combined are exothermic. Unconverted reactants can be used for the manufacture of other products, for example ammonium nitrate or sulfate, or they can be recycled for complete conversion to urea in a total-recycle process. Urea can be produced as prills, granules, pellets, crystals, and solutions. Solid urea is marketed as prills or granules. The advantage of prills is that, in general, they can be produced more cheaply than granules. Properties such as impact strength, crushing strength, and free-flowing behaviour are, in particular, important in product handling, storage, and bulk transportation. Typical impurities in the production are biuret and isocyanic acid: 2 NH2CONH2 → H2CONHCONH2 + NH3 NH2CONH2 → HNCO + NH3 The biuret content is a serious concern because it is often toxic to the very plants that are to be fertilized. Urea is classified on the basis of its biuret content.

Laboratory processes Ureas in the more general sense can be accessed in the laboratory by reaction of phosgene with primary or secondary amines, proceeding through an isocyanate intermediate. Non-symmetric ureas can be accessed by reaction of primary or secondary amines with an isocyanate.

Chemical properties Molecular and crystal structure The urea molecule is planar. In solid urea, the oxygen center is engaged in two N-H-O hydrogen bonds. The resulting dense and energetically favourable hydrogen-bond

network is probably established at the cost of efficient molecular packing: The structure is quite open, the ribbons forming tunnels with square cross-section. The carbon in urea is described as sp2 hybridized, the C-N bonds have significant double bond character, and the carbonyl oxygen is basic compared to, say, formaldehyde. Urea's high aqueous solubility reflects its ability to engage in extensive hydrogen bonding with water. By virtue of its tendency to form a porous frameworks, urea has the ability to trap many organic compounds. In these so-called clathrates, the organic "guest" molecules are held in channels formed by interpenetrating helices comprising of hydrogen-bonded urea molecules. This behaviour can be used to separate mixtures, e.g. in the production of aviation fuel and lubricating oils, and in the separation of paraffins. As the helices are interconnected, all helices in a crystal must have the same molecular handedness. This is determined when the crystal is nucleated and can thus be forced by seeding. The resulting crystals have been used to separate racemic mixtures.

Reactions Urea reacts with alcohols to form urethanes. Urea reacts with malonic esters to make barbituric acids.

Safety Urea can be irritating to skin, eyes, and the respiratory tract. Repeated or prolonged contact with urea in fertilizer form on the skin may cause dermatitis. High concentrations in the blood can be damaging. Ingestion of low concentrations of urea, such as are found in typical human urine, are not dangerous with additional water ingestion within a reasonable time-frame. Many animals (e.g., dogs) have a much more concentrated urine and it contains a higher urea amount than normal human urine; this can prove dangerous as a source of liquids for consumption in a life-threatening situation (such as in a desert). Urea can cause algal blooms to produce toxins, and its presence in the runoff from fertilized land may play a role in the increase of toxic blooms. The substance decomposes on heating above melting point, producing toxic gases, and reacts violently with strong oxidants, nitrites, inorganic chlorides, chlorites and perchlorates, causing fire and explosion.

Chapter- 7

Adenosine Triphosphate

Adenosine-5'-triphosphate (ATP) is a multifunctional nucleotide used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism. It is produced by photophosphorylation and cellular respiration and used by enzymes and structural proteins in many cellular processes, including biosynthetic reactions, motility, and cell division. One molecule of ATP contains three phosphate groups, and it is produced by ATP synthase from inorganic phosphate and adenosine diphosphate (ADP) or adenosine monophosphate (AMP). Metabolic processes that use ATP as an energy source convert it back into its precursors. ATP is therefore continuously recycled in organisms: the human body, which on average contains 250 grams (8.8 oz) of ATP turns over its own weight in ATP each day. ATP is used as a substrate in signal transduction pathways by kinases that phosphorylate proteins and lipids, as well as by adenylate cyclase, which uses ATP to produce the second messenger molecule cyclic AMP. The ratio between ATP and AMP is used as a way for a cell to sense how much energy is available and control the metabolic pathways that produce and consume ATP. Apart from its roles in energy metabolism and signaling,

ATP is also incorporated into nucleic acids by polymerases in the processes of DNA replication and transcription. The structure of this molecule consists of a purine base (adenine) attached to the 1' carbon atom of a pentose sugar (ribose). Three phosphate groups are attached at the 5' carbon atom of the pentose sugar. It is the addition and removal of these phosphate groups that inter-convert ATP, ADP and AMP. When ATP is used in DNA synthesis, the ribose sugar is first converted to deoxyribose by ribonucleotide reductase. ATP was discovered in 1929 by Karl Lohmann, but its correct structure was not determined until some years later. It was proposed to be the main energy-transfer molecule in the cell by Fritz Albert Lipmann in 1941. It was first artificially synthesized by Alexander Todd in 1948.

Physical and chemical properties ATP consists of adenosine — composed of an adenine ring and a ribose sugar — and three phosphate groups (triphosphate). The phosphoryl groups, starting with the group closest to the ribose, are referred to as the alpha (α), beta (β), and gamma (γ) phosphates. Consequently, as a nucleotide, it (and its relatives ADP and AMP) is basically a monomer of RNA. ATP is highly soluble in water and is quite stable in solutions between pH 6.8–7.4, but is rapidly hydrolysed at extreme pH. Consequently, ATP is best stored as an anhydrous salt. ATP is an unstable molecule in unbuffered water, in which it hydrolyses to ADP and phosphate. This is because the strength of the bonds between the phosphate groups in ATP are less than the strength of the hydrogen bonds (hydration bonds), between its products (ADP + phosphate), and water. Thus, if ATP and ADP are in chemical equilibrium in water, almost all of the ATP will eventually be converted to ADP. A system that is far from equilibrium contains Gibbs free energy, and is capable of doing work. Living cells maintain the ratio of ATP to ADP at a point ten orders of magnitude from equilibrium, with ATP concentrations a thousandfold higher than the concentration of ADP. This displacement from equilibrium means that the hydrolysis of ATP in the cell releases a large amount of free energy. Two high-energy phosphate bonds (phosphoanhydride bonds) (those that connect adjacent phosphates) in an ATP molecule are responsible for the high energy content of this molecule. In the context of biochemical reactions, these anhydride bonds are frequently—and sometimes controversially—referred to as high-energy bonds. Energy stored in ATP may be released upon hydrolysis of the anhydride bonds. The bonds formed after hydrolysis—or the phosphorylation of a residue by ATP—are lower in energy than the phosphoanhydride bonds of ATP. During enzyme-catalyzed hydrolysis of ATP or phosphorylation by ATP, the available free energy can be harnessed by a living system to do work.

Any unstable system of potentially reactive molecules could potentially serve as a way of storing free energy, if the cell maintained their concentration far from the equilibrium point of the reaction. However, as is the case with most polymeric biomolecules, the breakdown of RNA, DNA, and ATP into simpler monomers is driven by both energyrelease and entropy-increase considerations, in both standard concentrations, and also those concentrations encountered within the cell. The standard amount of energy released from hydrolysis of ATP can be calculated from the changes in energy under non-natural (standard) conditions, then correcting to biological concentrations. The net change in heat energy (enthalpy) at standard temperature and pressure of the decomposition of ATP into hydrated ADP and hydrated inorganic phosphate is −20.5 kJ/mol, with a change in free energy of 3.4 kJ/mol. The energy released by cleaving either a phosphate (Pi) or pyrophosphate (PPi) unit from ATP at standard state of 1 M are: ATP + H2O → ADP + Pi ΔG˚ = −30.5 kJ/mol (−7.3 kcal/mol) ATP + H2O → AMP + PPi ΔG˚ = −45.6 kJ/mol (−10.9 kcal/mol) These values can be used to calculate the change in energy under physiological conditions and the cellular ATP/ADP ratio. However, a more representative value (which takes AMP into consideration) called the Energy charge is increasingly being employed. The values given for the Gibbs free energy for this reaction are dependent on a number of factors, including overall ionic strength and the presence of alkaline earth metal ions such as Mg2+ and Ca2+. Under typical cellular conditions, ΔG is approximately −57 kJ/mol (−14 kcal/mol).

Ionization in biological systems ATP has multiple ionizable groups with different acid dissociation constants. In neutral solution, ATP is ionized and exists mostly as ATP4−, with a small proportion of ATP3−. As ATP has several negatively charged groups in neutral solution, it can chelate metals with very high affinity. The binding constant for various metal ions are (given as per mole) as Mg2+ (9 554), Na+ (13), Ca2+ (3 722), K+ (8), Sr2+ (1 381) and Li+ (25). Due to the strength of these interactions, ATP exists in the cell mostly in a complex with Mg2+.

Biosynthesis The ATP concentration inside the cell is typically 1–10 mM. ATP can be produced by redox reactions using simple and complex sugars (carbohydrates) or lipids as an energy source. For ATP to be synthesized from complex fuels, they first need to be broken down into their basic components. Carbohydrates are hydrolysed into simple sugars, such as glucose and fructose. Fats (triglycerides) are metabolised to give fatty acids and glycerol. The overall process of oxidizing glucose to carbon dioxide is known as cellular respiration and can produce about 30 molecules of ATP from a single molecule of glucose. ATP can be produced by a number of distinct cellular processes; the three main

pathways used to generate energy in eukaryotic organisms are glycolysis and the citric acid cycle/oxidative phosphorylation, both components of cellular respiration; and betaoxidation. The majority of this ATP production by a non-photosynthetic aerobic eukaryote takes place in the mitochondria, which can make up nearly 25% of the total volume of a typical cell.

Glycolysis In glycolysis, glucose and glycerol are metabolized to pyruvate via the glycolytic pathway. In most organisms, this process occurs in the cytosol, but in some protozoa such as the kinetoplastids, this is carried out in a specialized organelle called the glycosome. Glycolysis generates a net two molecules of ATP through substrate phosphorylation catalyzed by two enzymes: PGK and pyruvate kinase. Two molecules of NADH are also produced, which can be oxidized via the electron transport chain and result in the generation of additional ATP by ATP synthase. The pyruvate generated as an endproduct of glycolysis is a substrate for the Krebs Cycle.

Glucose In the mitochondrion, pyruvate is oxidized by the pyruvate dehydrogenase complex to Acetyl group, which is fully oxidized to carbon dioxide by the citric acid cycle (also known as the Krebs Cycle). Every "turn" of the citric acid cycle produces two molecules of carbon dioxide, one molecule of the ATP equivalent guanosine triphosphate (GTP) through substrate-level phosphorylation catalyzed by succinyl CoA synthetase, three molecules of the reduced coenzyme NADH, and one molecule of the reduced coenzyme FADH2. Both of these latter molecules are recycled to their oxidized states (NAD+ and FAD, respectively) via the electron transport chain, which generates additional ATP by oxidative phosphorylation. The oxidation of an NADH molecule results in the synthesis of between 2-3 ATP molecules, and the oxidation of one FADH2 yields between 1-2 ATP molecules. The majority of cellular ATP is generated by this process. Although the citric acid cycle itself does not involve molecular oxygen, it is an obligately aerobic process because O2 is needed to recycle the reduced NADH and FADH2 to their oxidized states. In the absence of oxygen the citric acid cycle will cease to function due to the lack of available NAD+ and FAD. The generation of ATP by the mitochondrion from cytosolic NADH relies on the malateaspartate shuttle (and to a lesser extent, the glycerol-phosphate shuttle) because the inner mitochondrial membrane is impermeable to NADH and NAD+. Instead of transferring the generated NADH, a malate dehydrogenase enzyme converts oxaloacetate to malate, which is translocated to the mitochondrial matrix. Another malate dehydrogenasecatalyzed reaction occurs in the opposite direction, producing oxaloacetate and NADH from the newly transported malate and the mitochondrion's interior store of NAD+. A transaminase converts the oxaloacetate to aspartate for transport back across the membrane and into the intermembrane space.

In oxidative phosphorylation, the passage of electrons from NADH and FADH2 through the electron transport chain powers the pumping of protons out of the mitochondrial matrix and into the intermembrane space. This creates a proton motive force that is the net effect of a pH gradient and an electric potential gradient across the inner mitochondrial membrane. Flow of protons down this potential gradient — that is, from the intermembrane space to the matrix — provides the driving force for ATP synthesis by ATP synthase. This enzyme contains a rotor subunit that physically rotates relative to the static portions of the protein during ATP synthesis. Most of the ATP synthesized in the mitochondria will be used for cellular processes in the cytosol; thus it must be exported from its site of synthesis in the mitochondrial matrix. The inner membrane contains an antiporter, the ADP/ATP translocase, which is an integral membrane protein used to exchange newly-synthesized ATP in the matrix for ADP in the intermembrane space. This translocase is driven by the membrane potential, as it results in the movement of about 4 negative charges out of the mitochondrial membrane in exchange for 3 negative charges moved inside. However, it is also necessary to transport phosphate into the mitochondrion; the phosphate carrier moves a proton in with each phosphate, partially dissipating the proton gradient.

Beta oxidation Fatty acids can also be broken down to acetyl-CoA by beta-oxidation. Each round of this cycle reduces the length of the acyl chain by two carbon atoms and produces one NADH and one FADH2 molecule, which are used to generate ATP by oxidative phosphorylation. Because NADH and FADH2 are energy-rich molecules, dozens of ATP molecules can be generated by the beta-oxidation of a single long acyl chain. The high energy yield of this process and the compact storage of fat explain why it is the most dense source of dietary calories.

Anaerobic respiration Anaerobic respiration or fermentation entails the generation of energy via the process of oxidation in the absence of O2 as an electron acceptor. In most eukaryotes, glucose is used as both an energy store and an electron donor. The equation for the oxidation of glucose to lactic acid is: C6H12O6

2CH3CH(OH)COOH + 2 ATP

In prokaryotes, multiple electron acceptors can be used in anaerobic respiration. These include nitrate, sulfate or carbon dioxide. These processes lead to the ecologicallyimportant processes of denitrification, sulfate reduction and acetogenesis, respectively.

ATP replenishment by nucleoside diphosphate kinases ATP can also be synthesized through several so-called "replenishment" reactions catalyzed by the enzyme families of nucleoside diphosphate kinases (NDKs), which use

other nucleoside triphosphates as a high-energy phosphate donor, and the ATP:guanidophosphotransferase family,

ATP production during photosynthesis In plants, ATP is synthesized in thylakoid membrane of the chloroplast during the lightdependent reactions of photosynthesis in a process called photophosphorylation. Here, light energy is used to pump protons across the chloroplast membrane. This produces a proton-motive force and this drives the ATP synthase, exactly as in oxidative phosphorylation. Some of the ATP produced in the chloroplasts is consumed in the Calvin cycle, which produces triose sugars.

ATP recycling The total quantity of ATP in the human body is about 0.1 mole. The majority of ATP is not usually synthesised de novo, but is generated from ADP by the aforementioned processes. Thus, at any given time, the total amount of ATP + ADP remains fairly constant. The energy used by human cells requires the hydrolysis of 100 to 150 moles of ATP daily which is around 50 to 75 kg. Typically, a human will use up their body weight of ATP over the course of the day. This means that each ATP molecule is recycled 1000 to 1500 times during a single day (100 / 0.1 = 1000). ATP cannot be stored, hence its consumption closely follows its synthesis.

Regulation of biosynthesis ATP production in an aerobic eukaryotic cell is tightly regulated by allosteric mechanisms, by feedback effects, and by the substrate concentration dependence of individual enzymes within the glycolysis and oxidative phosphorylation pathways. Key control points occur in enzymatic reactions that are so energetically favorable that they are effectively irreversible under physiological conditions. In glycolysis, hexokinase is directly inhibited by its product, glucose-6-phosphate, and pyruvate kinase is inhibited by ATP itself. The main control point for the glycolytic pathway is phosphofructokinase (PFK), which is allosterically inhibited by high concentrations of ATP and activated by high concentrations of AMP. The inhibition of PFK by ATP is unusual, since ATP is also a substrate in the reaction catalyzed by PFK; the biologically active form of the enzyme is a tetramer that exists in two possible conformations, only one of which binds the second substrate fructose-6-phosphate (F6P). The protein has two binding sites for ATP - the active site is accessible in either protein conformation, but ATP binding to the inhibitor site stabilizes the conformation that binds F6P poorly. A number of other small molecules can compensate for the ATP-induced shift in equilibrium conformation and reactivate PFK, including cyclic AMP, ammonium ions, inorganic phosphate, and fructose 1,6 and 2,6 biphosphate.

The citric acid cycle is regulated mainly by the availability of key substrates, particularly the ratio of NAD+ to NADH and the concentrations of calcium, inorganic phosphate, ATP, ADP, and AMP. Citrate - the molecule that gives its name to the cycle - is a feedback inhibitor of citrate synthase and also inhibits PFK, providing a direct link between the regulation of the citric acid cycle and glycolysis. In oxidative phosphorylation, the key control point is the reaction catalyzed by cytochrome c oxidase, which is regulated by the availability of its substrate—the reduced form of cytochrome c. The amount of reduced cytochrome c available is directly related to the amounts of other substrates:

which directly implies this equation:

Thus, a high ratio of [NADH] to [NAD+] or a low ratio of [ADP] [Pi] to [ATP] imply a high amount of reduced cytochrome c and a high level of cytochrome c oxidase activity. An additional level of regulation is introduced by the transport rates of ATP and NADH between the mitochondrial matrix and the cytoplasm.

Functions in cells Metabolism, synthesis, and active transport ATP is consumed in the cell by energy-requiring (endothermic) processes and can be generated by energy-releasing (exothermic) processes. In this way ATP transfers energy between spatially-separate metabolic reactions. ATP is the main energy source for the majority of cellular functions. This includes the synthesis of macromolecules, including DNA and RNA (see below), and proteins. ATP also plays a critical role in the transport of macromolecules across cell membranes, e.g. exocytosis and endocytosis.

Roles in cell structure and locomotion ATP is critically involved in maintaining cell structure by facilitating assembly and disassembly of elements of the cytoskeleton. In a related process, ATP is required for the shortening of actin and myosin filament crossbridges required for muscle contraction. This latter process is one of the main energy requirements of animals and is essential for locomotion and respiration.

Cell signalling

Extracellular signalling ATP is also a signalling molecule. ATP, ADP, or adenosine are recognised by purinergic receptors. Purinoreceptors might be the most abundant receptors in mammalian tissues (Abbracchio M.P. et al., 2008). In humans, this signalling role is important in both the central and peripheral nervous system. Activity-dependent release of ATP from synapses, axons and glia activates purinergic membrane receptors known as P2. The P2Y receptors are metabotropic, i.e. G protein-coupled and modulate mainly intracellular calcium and sometimes cyclic AMP levels. Though named between P2Y1 and P2Y15, only nine members of the P2Y family have been cloned, and some are only related through weak homology and several (P2Y5, P2Y7, P2Y9, P2Y10) do not function as receptors that raise cytosolic calcium. The P2X ionotropic receptor subgroup comprises seven members (P2X1–P2X7) which are ligandgated Ca2+-permeable ion channels that open when bound to an extracellular purine nucleotide. In contrast to P2 receptors (agonist order ATP > ADP > AMP > ADO), purinergic nucleotides like ATP are not strong agonists of P1 receptors which are strongly activated by adenosine and other nucleosides (ADO > AMP > ADP > ATP). P1 receptors have A1, A2a, A2b, and A3 subtypes ("A" as a remnant of old nomenclature of adenosine receptor), all of which are G protein-coupled receptors, A1 and A3 being coupled to Gi, and A2a and A2b being coupled to Gs. All adenosine receptors were shown to activate at least one subfamily of mitogen-activated protein kinases. The actions of adenosine are often antagonistic or synergistic to the actions of ATP. In the CNS, adenosine has multiple functions, such as modulation of neural development, neuron and glial signalling and the control of innate and adaptive immune systems (Abbracchio M.P. et al., 2008). Intracellular signalling ATP is critical in signal transduction processes. It is used by kinases as the source of phosphate groups in their phosphate transfer reactions. Kinase activity on substrates such as proteins or membrane lipids are a common form of signal transduction. Phosphorylation of a protein by a kinase can activate this cascade such as the mitogenactivated protein kinase cascade. ATP is also used by adenylate cyclase and is transformed to the second messenger molecule cyclic AMP, which is involved in triggering calcium signals by the release of calcium from intracellular stores. This form of signal transduction is particularly important in brain function, although it is involved in the regulation of a multitude of other cellular processes.

DNA and RNA synthesis In all known organisms, the deoxyribonucleotides that make up DNA are synthesized by the action of ribonucleotide reductase (RNR) enzymes on their corresponding ribonucleotides. These enzymes reduce the sugar residue from ribose to deoxyribose by

removing oxygen from the 2' hydroxyl group; the substrates are ribonucleoside diphosphates and the products deoxyribonucleoside diphosphates (the latter are denoted dADP, dCDP, dGDP, and dUDP respectively.) All ribonucleotide reductase enzymes use a common sulfhydryl radical mechanism reliant on reactive cysteine residues that oxidize to form disulfide bonds in the course of the reaction. RNR enzymes are recycled by reaction with thioredoxin or glutaredoxin. The regulation of RNR and related enzymes maintains a balance of dNTPs relative to each other and relative to NTPs in the cell. Very low dNTP concentration inhibits DNA synthesis and DNA repair and is lethal to the cell, while an abnormal ratio of dNTPs is mutagenic due to the increased likelihood of the DNA polymerase incorporating the wrong dNTP during DNA synthesis. Regulation of or differential specificity of RNR has been proposed as a mechanism for alterations in the relative sizes of intracellular dNTP pools under cellular stress such as hypoxia. In the synthesis of the nucleic acid RNA, ATP is one of the four nucleotides incorporated directly into RNA molecules by RNA polymerases. The energy driving this polymerization comes from cleaving off a pyrophosphate (two phosphate groups). The process is similar in DNA biosynthesis, except that ATP is reduced to the deoxyribonucleotide dATP, before incorporation into DNA.

Binding to proteins

An example of the Rossmann fold, a structural domain of a decarboxylase enzyme from the bacterium Staphylococcus epidermidis (PDB ID 1G5Q) with a bound flavin mononucleotide cofactor. Some proteins that bind ATP do so in a characteristic protein fold known as the Rossmann fold, which is a general nucleotide-binding structural domain that can also bind the cofactor NAD. The most common ATP-binding proteins, known as kinases, share a small number of common folds; the protein kinases, the largest kinase superfamily, all share common structural features specialized for ATP binding and phosphate transfer. ATP in complexes with proteins generally requires the presence of a divalent cation, almost always magnesium, which binds to the ATP phosphate groups. The presence of magnesium greatly decreases the dissociation constant of ATP from its protein binding partner without affecting the ability of the enzyme to catalyze its reaction once the ATP has bound. The presence of magnesium ions can serve as a mechanism for kinase regulation.

ATP analogues Biochemistry laboratories often use in vitro studies to explore ATP-dependent molecular processes. Enzyme inhibitors of ATP-dependent enzymes such as kinases are needed to examine the binding sites and transition states involved in ATP-dependent reactions. ATP analogs are also used in X-ray crystallography to determine a protein structure in complex with ATP, often together with other substrates. Most useful ATP analogs cannot be hydrolyzed as ATP would be; instead they trap the enzyme in a structure closely related to the ATP-bound state. Adenosine 5'-(gamma-thiotriphosphate) is an extremely common ATP analog in which one of the gamma-phosphate oxygens is replaced by a sulfur atom; this molecule is hydrolyzed at a dramatically slower rate than ATP itself and functions as an inhibitor of ATP-dependent processes. In crystallographic studies, hydrolysis transition states are modeled by the bound vanadate ion. However, caution is warranted in interpreting the results of experiments using ATP analogs, since some enzymes can hydrolyze them at appreciable rates at high concentration.

Chapter- 8

Other Biochemicals and Enzymes in Metabolic Biochemistry

Ribose D-Ribose

IUPAC name D-Ribose Identifiers CAS number 50-69-1 PubChem 5779 EC-number 200-059-4 Properties Molecular formula C5H10O5 Molar mass 150.13 g/mol Appearance white solid Melting point 95 °C, 368 K, 203 °F Solubility in water very soluble Chiral rotation [α]D −21.5º (H2O) Related compounds Arabinose Related aldopentoses Xylose

Related compounds

Lyxose Deoxyribose

Ribose is an organic compound with formula C5H10O5; specifically, a monosaccharide (simple sugar) with linear form H-(C=O)-(CHOH)4-H, which has all the hydroxyl groups on the same side in the Fischer projection. The term may refer to any of two enantiomers: almost always to D-ribose, which occurs widely in nature and is discussed here; or to its synthetic mirror image L-ribose, which is not found in nature and is of limited interest. D-ribose was first reported in 1891 by Emil Fischer. It is a C'-2 carbon enantiomer of the sugar D-arabinose (both isomers of which are named for their source, gum arabic) and

ribose itself is named as a transposition of the name of arabinose. Ribose comprises the backbone of RNA, a biopolymer that is the basis of genetic transcription. It is related to deoxyribose, as found in DNA. Once phosphorylated, ribose can become a subunit of ATP, NADH, and several other compounds that are critical to metabolism.

Structure Ribose is an aldopentose, that is a monosaccharide containing five carbon atoms that, in its open chain form, has an aldehyde functional group at one end. In the conventional numbering scheme for monosaccharides, the carbon atoms are numbered from C1' (in the aldehyde group) to C5'. The deoxyribose derivative, found in DNA, differs from ribose by having a hydrogen atom in place of the hydroxyl group in carbon C2'. Like many monosaccharides, ribose occurs in water as the linear form H-(C=O)(CHOH)4-H and any of two ring forms: ribofuranose ("C3'-endo"), with a fivemembered ring, and ribopyranose ("C2'-endo"), with a six-membered ring. The ribofuranose form is predominant in aqueous solution. The "D-" in the name D-ribose refers to the stereochemistry of the chiral carbon atom farthest away from the aldehyde group (C4'). In D-ribose, as in all D-sugars, this carbon atom has the same configuration as in D-glyceraldehyde.

Phosphorylation In biology, D-ribose must be phosphorylated by the cell before it can be used. Ribokinase catalyzes this reaction by converting D-ribose to D-ribose 5-phosphate. Once converted, D-ribose-5-phosphate is available for the manufacturing of the amino acids tryptophan and histidine, or for use in the pentose phosphate pathway. The absorption of D-ribose is 88-100% in the small intestines (up to 200 mg/kg/hr).

Marketed as a dietary supplement Ribose is marketed in the USA as a dietary supplement. Under US law, it is illegal to market dietary supplements as being safe and effective to treat a disease or condition. D-Ribose is marketed as a supplement for bodybuilders. Experiments have been done to

see if ribose actually does anything. Some studies have found no evidence that there is a benefit, while others have found increased endurance, recovery and muscle output in healthy individuals. As a supplement, ribose is often referred to as Corvalen, a brand name under which it was popularized. Ribose is also marketed as a stress relief ingredient.

Coenzyme A Coenzyme A

CAS number PubChem ChemSpider UNII MeSH Molecular formula Molar mass

Identifiers 85-61-0 6816 6557 SAA04E81UX Coenzyme+A Properties C21H36N7O16P3S 767.535

Coenzyme A (CoA, CoASH, or HSCoA) is a coenzyme, notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle. All sequenced genomes encode enzymes that use coenzyme A as a substrate, and

around 4% of cellular enzymes use it (or a thioester, such as acetyl-CoA) as a substrate. It is adapted from cysteamine, pantothenate, and adenosine triphosphate.

Biosynthesis Coenzyme A is synthesized in a five-step process from pantothenate and cysteine: 1. Pantothenate (vitamin B5) is phosphorylated to 4'-phosphopantothenate by the enzyme pantothenate kinase (PanK; CoaA; CoaX) 2. A cysteine is added to 4'-phosphopantothenate by the enzyme phosphopantothenoylcysteine synthetase (PPC-DC; CoaB) to form 4'-phospho-Npantothenoylcysteine (PPC) 3. PPC is decarboxylated to 4'-phosphopantetheine by phosphopantothenoylcysteine decarboxylase (CoaC) 4. 4'-phosphopantetheine is adenylylated to form dephospho-CoA by the enzyme phosphopantetheine adenylyl transferase (CoaD) 5. Finally, dephospho-CoA is phosphorylated using ATP to coenzyme A by the enzyme dephosphocoenzyme A kinase (CoaE).

Function Since coenzyme A is, in chemical terms, a thiol, it can react with carboxylic acids to form thioesters, thus functioning as an acyl group carrier. It assists in transferring fatty acids from the cytoplasm to mitochondria. A molecule of coenzyme A carrying an acetyl group is also referred to as acetyl-CoA. When it is not attached to an acyl group, it is usually referred to as 'CoASH' or 'HSCoA'. Coenzyme A is also the source of the phosphopantetheine group that is added as a prosthetic group to proteins such as acyl carrier protein and formyltetrahydrofolate dehydrogenase.

List of coenzyme A activated acyl groups • • • • •

• •

Acetyl-CoA Propionyl-CoA Acetoacetyl-CoA Coumaroyl-CoA (used in flavonoid and stilbenoid biosynthesis) Acyl derived from dicarboxylic acids o Malonyl-CoA o Succinyl-CoA o Hydroxymethylglutaryl-CoA (used in isoprenoid biosynthesis) o Pimelyl-CoA (used in biotin biosynthesis) Benzoyl CoA Butyryl CoA

Acetyl-CoA Acetyl coenzyme A or acetyl-CoA is an important molecule in metabolism, used in many biochemical reactions. Its main function is to convey the carbon atoms within the acetyl group to the citric acid cycle to be oxidized for energy production. In chemical structure, acetyl-CoA is the thioester between coenzyme A (a thiol) and acetic acid (an acyl group carrier). Acetyl-CoA is produced during the second step of aerobic cellular respiration, pyruvate decarboxylation, which occurs in the matrix of the mitochondria. Acetyl-CoA then enters the citric acid cycle. Acetyl-CoA is also an important component in the biogenic synthesis of the neurotransmitter acetylcholine. Choline, in combination with acetyl-CoA, is catalyzed by the enzyme choline acetyltransferase to produce acetylcholine and a coenzyme a byproduct.

Functions Pyruvate dehydrogenase and pyruvate formate lyase reactions The oxidative conversion of pyruvate into acetyl-CoA is referred to as the pyruvate dehydrogenase reaction. It is catalyzed by the pyruvate dehydrogenase complex. Other conversions between pyruvate and acetyl-CoA are possible. For example, pyruvate formate lyase disproportionates pyruvate into acetyl-CoA and formic acid. The pyruvate formate lyase reaction does not involve any net oxidation or reduction.

Fatty acid metabolism In animals, acetyl-CoA is essential to the balance between carbohydrate metabolism and fat metabolism. In normal circumstances, acetyl-CoA from fatty acid metabolism feeds into the citric acid cycle, contributing to the cell's energy supply. In the liver, when levels of circulating fatty acids are high, the production of acetyl-CoA from fat breakdown exceeds the cellular energy requirements. To make use of the energy available from the excess acetyl-CoA, ketone bodies are produced which can then circulate in the blood. Therefore, when at rest, both the skeletal and cardiac muscles satisfy their energy requirement mainly through oxidation of ketone bodies. In some circumstances, this can lead to the presence of very high levels of ketone bodies in the blood, a condition called ketosis. Benign dietary ketosis can safely occur in people following low-carbohydrate diets, which cause fats to be metabolised as a major source of energy. This is different from ketosis brought on as a result of starvation, and from ketoacidosis, a dangerous condition that can affect diabetics. In plants, de novo fatty acid synthesis occurs in the plastids. Many seeds accumulate large reservoirs of seed oils to support germination and early growth of the seedling

before it is a net photosynthetic organism. Fatty acids are incorporated into membrane lipids, the major component of most membranes.

Other reactions • • •

•

•

Two acetyl-CoA can be condensed to create acetoacetyl-CoA, the first step in the HMG-CoA/ mevalonic acid pathway leading to synthesis of isoprenoids. In animals HMG-CoA is a vital precursor to cholesterol and ketone synthesis. Acetyl-CoA is also the source of the acetyl group incorporated onto certain lysine residues of histone and non-histone proteins in the post-translational modification acetylation, a reaction catalyzed by acetyltransferases. In plants and animals, cytosolic acetyl-CoA is synthesized by ATP citrate lyase . When glucose is abundant in the blood of animals, it is converted via glycolysis in the cytosol to pyruvate, and thence to acetyl-CoA in the mitochondrion. The excess of acetyl-CoA results in production of excess citrate, which is exported into the cytosol to give rise to cytosolic acetyl-CoA. Acetyl-CoA can be carboxylated in the cytosol by acetyl-CoA carboxylase, giving rise to malonyl-CoA, a substrate required for synthesis of flavonoids and related polyketides, for elongation of fatty acids to produce waxes, cuticle, and seed oils in members of the Brassica family, and for malonation of proteins and other phytochemicals . In plants, these include sesquiterpenes, brassinosteroids (hormones), and membrane sterols.

Interactive pathway map

Propionyl-CoA Propionyl-CoA is a coenzyme A derivative of propionic acid.

Metabolism in animals Production There are several different ways in which it is formed: • •

It is formed as a product of beta-oxidation of odd-chain fatty acids, which occur in meat of ruminant animals. It is also a product of metabolism of isoleucine and valine.

•

It is a product of alpha-ketobutyric acid, which in turn is a product of digestion of threonine and methionine.

Metabolic fate In mammals, propionyl-CoA is converted to (S)-methylmalonyl-CoA by propionyl-CoA carboxylase, a biotin-dependent enzyme also requiring bicarbonate and ATP. This product is converted to (R)-methylmalonyl-CoA by methylmalonyl-CoA racemase. (R)-Methylmalonyl-CoA is converted to succinyl-CoA by methylmalonyl-CoA mutase, an enzyme requiring cobalamin to catalyze the carbon-carbon bond migration. The methylmalonyl-CoA mutase mechanism begins with the cleavage of the bond between the 5' CH2- of 5'-deoxyadenosyl and the cobalt, which is in its 3+ oxidation state (III), which produces a 5'-deoxyadenosyl radical and cabalamin in the reduced Co(II) oxidation state. Next, this radical abstracts a hydrogen atom from the methyl group of methylmalonylCoA, which generates a methylmalonyl-CoA radical. It is believed that this radical forms a carbon-cobalt bond to the coenzyme which is then followed by the rearrangement of the substrate's carbon skeleton, thus producing a succinyl-CoA radical. This radical then goes on to abstract a hydrogen from the previously produced 5'-deoxyadenosine, again creating a deoxyadenosyl radical, which attacks the coenzyme to reform the initial complex. A defect in methylmalonyl-CoA mutase enzyme results in methylmalonic aciduria, a dangerous disorder that causes a lowering of blood pH.

Metabolism in plants and insects In plants and insects propionyl-CoA is metabolized to acetate in a very different way, similar to beta oxidation. Not all details of this pathway have been worked out, but it appears to involve formation of acrylyl-CoA, then 3-hydroxypropionyl-CoA. This is metabolized with loss of carbon 1 of 3-hydroxypropionyl-CoA as carbon dioxide, while carbon 3 becomes carbon 1 of acetate.

Additional images

Coenzyme A