Biotechnology - Biochemistry

Biotechnology - Biochemistry

Oxidation of fatty acids

Fatty acid oxidation is the mitochondrial aerobic process of breaking down a fatty acid into acetyl-CoA units. Fatty acid oxidation and production of acetyl-CoA is a central energy yielding pathway in many organisms. In mammals, oxidation of fatty acids in heart and liver provides most of the energetic needs under all physiological circumstances. Fatty acid contains a long hydrocarbon chain and a terminal carboxylate group. Fatty acids are having major physiological roles, i.e., they are building blocks of phospholipids and glycolipids. Many proteins are modified by the covalent attachment of fatty acids, which targets them to membrane locations. Fatty acids are stored as triacylglycerols (also called neutral fats or triglycerides), which are uncharged esters of fatty acids with glycerol. Fatty acid derivatives serve as hormones and intracellular messenger. The electrons removed from fatty acids during oxidation pass through the respiratory chain, driving ATP synthesis. Although the biological role of fatty acid oxidation differs from organism to organism, the mechanism is the same. 

Beta-oxidation of fatty acid is the catabolic process by which fatty acid molecules are broken down in the cytosol in prokaryotes and in the mitochondria in eukaryotes to generate acetyl-CoA, which enters the citric acid cycle. The co-enzymes NADH and FADH2 are co-enzymes used in the electron transport chain. It is named as such because the beta carbon of the fatty acid undergoes oxidation to a carbonyl group.

TCA cycle

The citric acid cycle (CAC) – also known as the TCA cycle (tricarboxylic acid cycle) or the Krebs cycle – is a series of chemical reactions used by all aerobic organisms to release stored energy through the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins. The citric acid cycle is a hub in metabolism, with catabolic pathways leading in and anabolic pathways leading out, and it is closely regulated in co-ordination with other pathways. The starting material for the citric acid cycle is acetyl-CoA  produced from pyruvate, the product of glycolysis by the action of pyruvate dehydrogenase complex. The TCA cycle consists of eight steps catalyzed by eight different enzymes. Each complete turn of the cycle results in the regeneration of oxaloacetate and the formation of two molecules of carbon dioxide.

Oxidation of Amino Acids

The fraction of  metabolic energy obtained from amino acids, whether they are derived from dietary protein or from tissue protein, varies greatly with the type of organism and with metabolic conditions. In animals, amino acids undergo oxidative degradation in three different metabolic circumstances. The first one is during the normal synthesis and degradation of cellular proteins, some amino acids that are released from protein breakdown and are not needed for new protein synthesis undergo oxidative degradation. The second one, is during when a diet is rich in protein and the ingested amino acids exceed the body's need for protein synthesis, the extra is catabolized and the amino acids can't be stored. The third circumstance is during starvation or during uncontrolled diabetes mellitus, when carbohydrates are not properly utilized or unavailable, cellular proteins are used as fuel. Amino acids loose their amino groups to form α-keto acids and the  α-keto acids undergo oxidation to produce carbon dioxide and water or three and four carbon units that can be converted to glucose. Every amino acid contains an amino group, and in the pathways of amino acid degradation, in one step, the α-amino group is separated from the carbon skeleton and shunted into the pathways of amino group metabolism.

Nitrogen excretion and the Urea cycle

When amino acids are catabolized, ammonia is formed as a by-product. Ammonia is toxic to most animals at relatively low concentrations, and can interrupt nerve conduction and alter metabolism of amino acid neurotransmitters, fats, carbohydrates, and ATP . Most terrestrial animals are ureotelic, excreting amino nitrogen in the form of urea, birds and reptiles are uricotelic, excreting amino nitrogen as uric acid. Plants recycle virtually all amino groups, and nitrogen excretion occurs under very unusual circumstances. In ureotelic organisms, the ammonia deposited in the mitochondria of hepatocytes and is converted to urea in the urea cycle. Liver produces urea, which passes into the bloodstream and thus to the kidneys and is excreted in the urine.

Oxidative phosphorylation

Oxidative phosphorylation is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing the chemical energy stored within in order to produce adenosine triphosphate (ATP). In eukaryotes, Oxidative phosphorylation occurs in mitochondria and it involves the reduction of oxygen to water with electrons donated by NADH and FADH2. All aerobic organisms carry out oxidative phosphorylation. The flow of electrons from NADH or FADH2 to oxygen through protein complexes located in the mitochondrial inner membrane leads to the pumping of protons out of the mitochondrial matrix. The resulting uneven distribution of protons generates a pH gradient and a transmembrane electrical potential that creates a proton-motive force. ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex.

Photophosphorylation

Photophosphorylation is the process of utilizing light energy from photosynthesis to convert ADP to ATP. In eukaryotes, photophosphorylation occurs in chloroplast and it involves the oxidation of water to oxygen, with NADP+ as the electron acceptor. In photophosphorylation, electrons flow through a series of membrane-bound carriers including cytochromes, quinones, and iron-sulfur proteins, while protons are pumped across a membrane to create an electrochemical potential. In plants, photosynthesis includes two processes: the light reactions, which occur only during day time and the dark reactions, which are driven by products of the light reactions.

Biosynthesis of Carbohydrate in plants and bacteria

Plants are autotrophs and can convert inorganic carbon (carbon dioxide) into organic compounds. Carbohydrate biosynthesis occurs primarily in plastids (membrane bound organelles in plants) and the movement of intermediates between cellular compartments is an important aspect of metabolism. Plants have thick cell walls made of carbohydrate polymers which constitute a significant proportion of the cell's carbohydrate. Plants and photosynthetic microorganisms can synthesize carbohydrates from carbon dioxide and water, reducing carbon dioxide  at the expense of the energy and reducing power furnished by the ATP and NADPH which are generated in the light reactions. Carbon dioxide is assimilated through a cyclic pathway, which was discovered by Melvin Calvin, Andrew Benson and James A. Bassham and is known as Calvin cycle.

Photorespiration is the process of light-dependent uptake of molecular oxygen (O2) concomitant with release of carbon dioxide (CO2) from organic compounds. Photorespiration refers to a process in plant metabolism where the enzyme RuBisCO oxygenates RuBP, wasting some of the energy produced by photosynthesis. In C4 plants, the carbon-assimilation pathway minimizes photorespiration, carbon dioxide is first fixed in mesophyll cells into a four-carbon compound which passes into bundle-sheath cells and releases carbon dioxide in high concentrations. The enzyme RuBisCO fixes the released carbon dioxide and the remaining reactions of the Calvin cycle occur as in C3 plants. In CAM plants, carbon dioxide is fixed malate in the dark and stored in vacuoles.

The excess carbohydrate produced by plants in bright light, is converted to sucrose and transported to other parts of the plant, to be used as fuel or stored. The synthesis of starch and sucrose occurs in different cellular compartments and these processes are coordinated by a variety of regulatory mechanisms that respond to changes in light level and photosynthetic rate. ADP-glucose is the substrate for strach synthesis in plant plastids and for glycogen synthesis in bacteria.

Biosynthesis of Lipids

Lipids are the principal form of stored energy in most organisms and major constituents of cellular membranes. The ability to synthesize a variety of lipids is essential to all organisms. Fatty acid synthesis is the creation of fatty acids from acetyl-CoA and NADPH through the action of enzymes called fatty acid synthases. This process takes place in the cytoplasm of the cell. Fatty acid biosynthesis requires the participation of a three carbon intermediate, malonyl-CoA, which is not involved in fatty acid breakdown. The synthesis of malonyl-CoA from acetyl-CoA is catalyzed by the enzyme acetyl-CoA carboxylase in an irreversible process.  The long carbon chains of fatty acids are assembled in a repeating four step sequence.  Each malonyl group and acetyl group is activated by a thioester that links it to fatty acid synthase (multi enzyme complex).  Condensation of an acetyl group from acetyl-CoA (activated acyl group)  and two carbon atoms derived from malonyl-CoA, with elimination of carbon dioxide from the malonyl group, extends the acyl chain by two carbons. When the chain length reaches 16 carbons, the product leaves the cycle. The four-step process of fatty acid synthesis is the same in all organisms.

Most of the fatty acids synthesized by an organism either get incorporated into triacylglycerols for the storage of metabolic energy or get incorporated into the phospholipid components of membranes. Animals can synthesize and store large quantities of triacylglycerols, to be used later as fuel. Triacylglycerols are formed by the reaction of two molecules of fatty acyl-CoA with glycerol 3-phosphate to form phosphatidic acid, which is dephosphorylated to a diacylglycerol and then acylated by a third molecule of fatty-acyl CoA to yield a triacylglycerol. The rate of triacylglycerol biosynthesis is altered by the action of several hormones.

In eukaryotic cells, phospholipid synthesis occurs primarily on the surfaces of smooth endoplasmic reticulum and the mitochondrial inner membrane. The principal precursors of glycerophospholipids are diacylglycerols. Membrane lipids are insoluble in water, so they can't simply diffuse from the endoplasmic reticulum to their point of insertion. So they are delivered in membrane vesicles that bud from the Golgi complex then move to and fuse with the target membrane.

Cholesterol is an essential molecule in many animals, including humans but is not required in the mammalian diet. Cholesterol plays an important role as a component of cellular membranes and as a precursor of steroid hormones and bile acids. Biosynthesis of cholesterol generally takes place in the endoplasmic reticulum of hepatic cells and begins with acetyl- CoA, which is mainly derived from an oxidation reaction in the mitochondria. Cholesterol is essential for all animal life, with each cell capable of synthesizing it by way of a complex 37-step process. This begins with the mevalonate or HMG-CoA reductase pathway, the target of statin drugs, which encompasses the first 18 steps. This is followed by 19 additional steps to convert the resulting lanosterol into cholesterol. The steroid hormones glucocorticoids, mineralcorticoids and sex hormones are produced  from cholesterol by alteration of the side chain and introduction of oxygen atoms into the steroid ring system.

Biosynthesis of amino acids and nucleotides

Amino acids and nucleotides are charged molecules, so their levels must be regulated to maintain electrochemical balance in the  cell. The biosynthetic pathways for amino acids and nucleotides share a requirement for nitrogen. The nitrogen cycle maintains a pool of biologically available nitrogen. Air is the most important source of nitrogen but conversion of atmospheric nitrogen into useful forms for living organisms is done by relatively few species. Atmospheric nitrogen is fixed by nitrogen-fixing bacteria to produce ammonia is the first step in the  nitrogen cycle. Soil bacteria oxidize ammonia to nitrite (NO2-) and nitrite to nitrate (NO3-) and the process is known as nitrification. Plants and many bacteria can take up and readily reduce nitrate and nitrite through the action of nitrate and nitrite reductases. The ammonia so formed is incorporated into amino acids by plants. Soil bacteria maintain a balance between fixed nitrogen and atmospheric nitrogen by converting nitrate to N2 (nitrogen), the process is called dentrification. Biological nitrogen fixation is carried out by a highly conserved complex of proteins called the nitrogenase complex. In living systems, reduced nitrogen is incorporated first into amino acids and then into other biomolecules, including nucleotides. Glutamate and glutamine are the nitrogen donors, in different biosynthetic reactions. Glutamine synthase catalyzes the synthesis of glutamine from glutamate.

Plants and bacteria synthesize all twenty common amino acids. Out of the twenty basic amino acids, humans are unable to synthesize eight amino acids. Amino acids that must be obtained from the diet are called essential amino acids. Nonessential amino acids are produced in the body, and can be synthesized in simple pathways In addition, the amino acids arginine, cysteine, glycine, glutamine, histidine, proline, serine, and tyrosine are considered conditionally essential, meaning they are not normally required in the diet but must be supplied exogenously to specific populations that do not synthesize it in adequate amounts.  The regulation of the synthesis of glutamate from α-ketoglutarate is subject to regulatory control of the Citric Acid Cycle as well as mass action dependent on the concentrations of reactants involved due to the reversible nature of the transamination and glutamate dehydrogenase reactions. The conversion of glutamate to glutamine is regulated by glutamine synthetase (GS) and is a key step in nitrogen metabolism. The amino acid biosynthetic pathways are subject to allosteric end-product inhibition; the regulatory enzyme is usually the first in the sequence. 

Many important biomolecules are derived from amino acids. Glycine is a precursor of porphyrins. Glutathione is an important cellular reducing agent formed from three amino acids. Many plant substances are produced by aromatic amino acids. Nitric oxide, a biological messenger is produced from arginine.

Nucleotides are the precursors of nucleic acids (DNA and RNA). They are essential carriers of chemical energy. There are two pathways for nucleotide biosynthesis i.e., denovo pathways and salvage pathways. The denovo pathways are nearly identical in all living organisms. The purine ring system is built up step by step beginning with 5-phosphoribosylamine. The amino acids glutamine, glycine and aspartate furnish all the nitrogen atoms of purines. Pyrimidines are synthesized from carbamoyl phosphate and aspartate, and the attachment of ribose-5-phosphate yields the pyrimidine ribonucleotides. Uric acid and urea are the end products of purine and pyrimidine degradation.

Hormones and their regulation

An essential characteristic of multicellular organisms is cell differentiation and division of labor. The specialized functions of the tissues and organs of complex organisms impose characteristic fuel requirements and patterns of metabolism. Hormonal signals integrate and coordinate the metabolic activities of different tissues and optimize the allocation of fuels and precursors to each organ. Hormones are chemical messengers that are secreted directly into the blood, which carries them to organs and tissues of the body to exert their functions. Hormones are required for the correct development of both animals and plants. Hormones affect distant cells by binding to specific receptor proteins in the target cell, resulting in a change in cell function. When a hormone binds to the receptor, it results in the activation of a signal transduction pathway that typically activates gene transcription, resulting in increased expression of target proteins. Mammals have several classes of hormones, distinguishable by their chemical structures and their modes of action. Some hormones such as peptide, amine, and eicosanoid hormones act from outside the target cells through surface receptors and hormones like steroid, vitamin D, retinoid and thyroid act through nuclear receptors. Insulin signals many body tissues whether the blood glucose is higher than necessary, so that cells take up excess glucose from the blood and convert it to glycogen and triacylglycerol. Low blood glucose is signaled by glucagon and tissues respond by producing glucose through glycogen breakdown and by oxidizing fats to reduce the use of glucose.

Multiple Choice Questions with explanations

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• Biochemistry - Multiple Choice Questions

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Level: Intermediate Level

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Instructor: Swagatika Sahu