Life Sciences Fundamentals and Practice PDF

Life Sciences Fundamentals and Practice Part – I Fourth edition Pranav Kumar Former faculty, Department of Biotechnology Jamia Millia Islamia, New Delhi, India Usha Mina Scientist, Division of Environmental Sciences Indian Agricultural Research Institute (IARI), New Delhi, India Pathfinder Publ...

Life Sciences Fundamentals and Practice Part – I Fourth edition Pranav Kumar Former faculty, Department of Biotechnology Jamia Millia Islamia, New Delhi, India Usha Mina Scientist, Division of Environmental Sciences Indian Agricultural Research Institute (IARI), New Delhi, India Pathfinder Publication New Delhi, India  Pranav Kumar Former faculty, Department of Biotechnology Jamia Millia Islamia, New Delhi, India Usha Mina Scientist, Division of Environmental Sciences Indian Agricultural Research Institute (IARI), New Delhi, India Life Sciences Fundamentals and Practice, Fourth edition ISBN: 978-81-906427-0-5 (paperback) Copyright © 2014 by Pathfinder Publication, all rights reserved. This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reproduced by any mechanical, photographic, or electronic process, or in the form of a phonographic recording, nor it may be stored in a retrieval system, transmitted, or otherwise copied for public or private use, without written permission from the publisher. Publisher : Pathfinder Publication Production editor : Ajay Kumar Copy editor : Jomesh Joseph Illustration and layout : Pradeep Verma Cover design : Pradeep Verma Marketing director : Arun Kumar Production coordinator : Murari Kumar Singh Printer : Ronit Enterprises, New Delhi, India Pathfinder Publication A unit of Pathfinder Academy Private Limited, New Delhi, India. www.thepathfinder.in 09350208235 Preface Life Sciences have always been a fundamental area of science. The exponential increase in the quantity of scientific information and the rate, at which new discoveries are made, require very elaborate, interdisciplinary and up-to-date information and their understanding. This fourth edition of Life sciences, Fundamentals and practice includes extensive revisions of the previous edition. We have attempted to provide an extraordinarily large amount of information from the enormous and ever-growing field in an easily retrievable form. It is written in clear and concise language to enhance self-motivation and strategic learning skill of the students and empowering them with a mechanism to measure and analyze their abilities and the confidence of winning. We have given equal importance to text and illustrations. The fourth edition has a number of new figures to enhance understanding. At the same time, we avoid excess detail, which can obscure the main point of the figure. We have retained the design elements that have evolved through the previous editions to make the book easier to read. Sincere efforts have been made to support textual clarifications and explanations with the help of flow charts, figures and tables to make learning easy and convincing. The chapters have been supplemented with self-tests and questions so as to check one’s own level of understanding. Although the chapters of this book can be read independently of one another, they are arranged in a logical sequence. Each page is carefully laid out to place related text, figures and tables near one another, minimizing the need for page turning while reading a topic. I have given equal importance to text and illustrations as well. We hope you will find this book interesting, relevant and challenging. Acknowledgements Our students were the original inspiration for the first edition of this book, and we remain continually grateful to them, because we learn from them how to think about the life sciences, and how to communicate knowledge in most meaningful way. We thank, Dr. Diwakar Kumar Singh and Mr. Ajay Kumar, reviewers of this book, whose comment and suggestions were invaluable in improving the text. Any book of this kind requires meticulous and painstaking efforts by all its contributors. Several diligent and hardworking minds have come together to bring out this book in this complete form. We are much beholden to each of them and especially to Dr. Neeraj Tiwari. This book is a team effort, and producing it would be impossible without the outstanding people of Pathfinder Publication. It was a pleasure to work with many other dedicated and creative people of Pathfinder Publication during the production of this book, especially Pradeep Verma. Pranav Kumar Usha Mina iii This is a preview. The total pages displayed will be limited. Contents Chapter 1 Biomolecules and Catalysis 1.1 Amino acids and Proteins 1 1.1.1 Optical properties 2 1.1.2 Absolute configuration 4 1.1.3 Standard and non-standard amino acids 5 1.1.4 Titration of amino acids 8 1.1.5 Peptide and polypeptide 11 1.1.6 Peptide bond 12 1.1.7 Protein structure 14 1.1.8 Denaturation of proteins 18 1.1.9 Solubilities of proteins 19 1.1.10 Simple and conjugated proteins 20 1.2 Fibrous and globular proteins 20 1.2.1 Collagen 21 1.2.2 Elastin 22 1.2.3 Keratins 23 1.2.4 Myoglobin 23 1.2.5 Hemoglobin 25 1.2.6 Models for the behavior of allosteric proteins 29 1.3 Protein folding 31 1.3.1 Molecular chaperones 32 1.3.2 Amyloid 33 1.4 Protein sequencing and assays 34 1.5 Nucleic acids 42 1.5.1 Nucleotides 42 1.5.2 Chargaff’s rules 46 1.6 Structure of dsDNA 47 1.6.1 B-DNA 47 1.6.2 Z-DNA 49 1.6.3 Triplex DNA 49 1.6.4 G-quadruplex 50 1.6.5 Stability of the double helical structure of DNA 51 1.6.6 Thermal denaturation 51 1.6.7 Quantification of nucleic acids 53 1.6.8 Supercoiled forms of DNA 53 1.6.9 DNA: A genetic material 56 v 1.7 RNA 58 1.7.1 Alkali-catalyzed cleavage of RNA 60 1.7.2 RNA world hypothesis 61 1.7.3 RNA as genetic material 61 1.8 Carbohydrates 63 1.8.1 Monosaccharide 63 1.8.2 Epimers 64 1.8.3 Cyclic forms 65 1.8.4 Derivatives of monosaccharide 67 1.8.5 Disaccharides and glycosidic bond 68 1.8.6 Polysaccharides 70 1.8.7 Glycoproteins 72 1.8.8 Reducing and non-reducing sugar 73 1.9 Lipids 73 1.9.1 Fatty acids 74 1.9.2 Triacylglycerol and Wax 75 1.9.3 Phospholipids 76 1.9.4 Glycolipids 78 1.9.5 Steroid 79 1.9.6 Eicosanoid 79 1.9.7 Plasma lipoproteins 81 1.10 Vitamins 82 1.10.1 Water-soluble vitamins 82 1.10.2 Fat-soluble vitamins 86 1.11 Enzymes 89 1.11.1 Naming and classification of enzyme 90 1.11.2 What enzyme does? 91 1.11.3 How enzymes operate? 92 1.11.4 Enzyme kinetics 94 1.11.5 Enzyme inhibition 102 1.11.6 Regulatory enzymes 105 1.11.7 Isozymes 106 1.11.8 Zymogen 107 1.11.9 Ribozyme 108 1.11.10 Examples of enzymatic reactions 108 Chapter 2 Bioenergetics and Metabolism 2.1 Bioenergetics 117 2.2 Metabolism 122 2.3 Respiration 123 2.3.1 Aerobic respiration 123 2.3.2 Glycolysis 124 2.3.3 Pyruvate oxidation 129 vi  2.3.4 Krebs cycle 131 2.3.5 Anaplerotic reaction 134 2.3.6 Oxidative phosphorylation 135 2.3.7 Inhibitors of electron transport 139 2.3.8 Electrochemical proton gradient 140 2.3.9 Chemiosmotic theory 141 2.3.10 ATP synthase 142 2.3.11 Uncoupling agents and ionophores 144 2.3.12 ATP-ADP exchange across the inner mitochondrial membrane 144 2.3.13 Shuttle systems 145 2.3.14 P/O ratio 147 2.3.15 Fermentation 148 2.3.16 Pasteur effect 150 2.3.17 Warburg effect 150 2.3.18 Respiratory quotient 151 2.4 Glyoxylate cycle 151 2.5 Pentose phosphate pathway 152 2.6 Entner-Doudoroff pathway 154 2.7 Photosynthesis 154 2.7.1 Photosynthetic pigment 155 2.7.2 Absorption and action spectra 158 2.7.3 Fate of light energy absorbed by photosynthetic pigments 160 2.7.4 Concept of photosynthetic unit 161 2.7.5 Hill reaction 162 2.7.6 Oxygenic and anoxygenic photosynthesis 162 2.7.7 Concept of pigment system 163 2.7.8 Stages of photosynthesis 165 2.7.9 Light reactions 165 2.7.10 Prokaryotic photosynthesis 171 2.7.11 Non-chlorophyll based photosynthesis 173 2.7.12 Dark reaction: Calvin cycle 174 2.7.13 Starch and sucrose synthesis 177 2.8 Photorespiration 178 2.8.1 C4 cycle 179 2.8.2 CAM pathway 180 2.9 Carbohydrate metabolism 182 2.9.1 Gluconeogenesis 182 2.9.2 Glycogen metabolism 187 2.10 Lipid metabolism 192 2.10.1 Synthesis and storage of triacylglycerols 192 2.10.2 Biosynthesis of fatty acid 194 2.10.3 Fatty acid oxidation 198 2.10.4 Biosynthesis of cholesterol 205 2.10.5 Steroid hormones and Bile acids 206 vii 2.11 Amino acid metabolism 208 2.11.1 Amino acid synthesis 208 2.11.2 Amino acid catabolism 211 2.11.3 Molecules derived from amino acids 217 2.12 Nucleotide metabolism 218 2.12.1 Nucleotide synthesis 218 2.12.2 Nucleotide degradation 225 Chapter 3 Cell Structure and Functions 3.1 What is a Cell? 231 3.2 Structure of eukaryotic cells 232 3.2.1 Plasma membrane 232 3.2.2 ABO blood group 239 3.2.3 Transport across plasma membrane 241 3.3 Membrane potential 248 3.4 Transport of macromolecules across plasma membrane 258 3.4.1 Endocytosis 258 3.4.2 Fate of receptor 262 3.4.3 Exocytosis 263 3.5 Ribosome 264 3.5.1 Protein targeting and translocation 265 3.6 Endoplasmic reticulum 266 3.6.1 Endomembrane system 271 3.6.2 Transport of proteins across the ER membrane 271 3.6.3 Transport of proteins from ER to cis Golgi 276 3.7 Golgi complex 277 3.7.1 Transport of proteins through cisternae 279 3.7.2 Transport of proteins from the TGN to lysosomes 280 3.8 Vesicle fusion 281 3.9 Lysosome 282 3.10 Vacuoles 284 3.11 Mitochondria 284 3.12 Plastids 287 3.13 Peroxisome 288 3.14 Cytoskeleton 289 3.14.1 Microtubules 289 3.14.2 Kinesins and Dyneins 292 3.14.3 Cilia and Flagella 292 3.14.4 Centriole 295 3.14.5 Actin filament 295 3.14.6 Myosin 297 3.14.7 Muscle contraction 298 3.14.8 Intermediate filaments 302 viii 3.15 Cell junctions 303 3.16 Cell adhesion molecules 306 3.17 Extracellular matrix of animals 307 3.18 Plant cell wall 308 3.19 Nucleus 310 3.20 Cell signaling 313 3.20.1 Signal molecules 314 3.20.2 Receptors 315 3.20.3 GPCR and G-proteins 317 3.20.4 Ion channel-linked receptors 326 3.20.5 Enzyme-linked receptors 326 3.20.6 Nitric oxide 332 3.20.7 Two-component signaling systems 333 3.20.8 Chemotaxis in bacteria 334 3.20.9 Quorum sensing 335 3.20.10 Scatchard plot 336 3.21 Cell Cycle 338 3.21.1 Role of Rb protein in cell cycle regulation 342 3.21.2 Role of p53 protein in cell cycle regulation 343 3.21.3 Replicative senescence 344 3.22 Mechanics of cell division 344 3.22.1 Mitosis 344 3.22.2 Meiosis 351 3.22.3 Nondisjunction and aneuploidy 357 3.23 Apoptosis 358 3.24 Cancer 361 3.25 Stem cells 368 Chapter 4 Prokaryotes and Viruses 4.1 General features of Prokaryotes 373 4.2 Phylogenetic overview 374 4.3 Structure of bacterial cell 374 4.4 Bacterial genome : Bacterial chromosome and plasmid 385 4.5 Bacterial nutrition 389 4.5.1 Culture media 391 4.5.2 Bacterial growth 391 4.6 Horizontal gene transfer and genetic recombination 395 4.6.1 Transformation 396 4.6.2 Transduction 398 4.6.3 Conjugation 402 4.7 Bacterial taxonomy 407 4.8 General features of important bacterial groups 409 4.9 Archaebacteria 411 ix 4.10 Bacterial toxins 412 4.11 Control of microbial growth 414 4.12 Virus 418 4.12.1 Bacteriophage (Bacterial virus) 419 4.12.2 Life cycle of bacteriophage 420 4.12.3 Plaque assay 423 4.12.4 Genetic analysis of phage 425 4.12.5 Animal viruses 428 4.12.6 Plant viruses 438 4.13 Prions and Viroid 439 4.13.1 Bacterial and viral disease 440 Chapter 5 Immunology 5.1 Innate immunity 443 5.2 Adaptive immunity 445 5.3 Cells of the immune system 447 5.3.1 Lymphoid progenitor 448 5.3.2 Myeloid progenitor 450 5.4 Organs involved in the adaptive immune response 451 5.4.1 Primary lymphoid organs 451 5.4.2 Secondary lymphoid organs/tissues 452 5.5 Antigens 453 5.6 Major-histocompatibility complex 457 5.6.1 MHC molecules and antigen presentation 459 5.6.2 Antigen processing and presentation 460 5.6.3 Laboratory mice 462 5.7 Immunoglobulins : Structure and function 463 5.7.1 Basic structure of antibody molecule 463 5.7.2 Different classes of immunoglobulin 466 5.7.3 Action of antibody 468 5.7.4 Antigenic determinants on immunoglobulins 468 5.8 B-cell maturation and activation 470 5.9 Kinetics of the antibody response 476 5.10 Monoclonal antibodies and Hybridoma technology 477 5.10.1 Engineered monoclonal antibodies 478 5.11 Organization and expression of Ig genes 480 5.12 Generation of antibody diversity 486 5.13 T-cells and CMI 489 5.13.1 Superantigens 499 5.14 Cytokines 500 5.15 The complement system 504 5.16 Hypersensitivity 507 5.17 Autoimmunity 510 5.18 Transplantation 510 x 5.19 Immunodeficiency diseases 511 5.20 Failures of host defense mechanisms 511 5.21 Vaccines 513 Chapter 6 Diversity of Life 6.1 Taxonomy 520 6.1.1 Nomenclature 520 6.1.2 Classification 521 6.1.3 Biological species concept 521 6.1.4 Phenetics 522 6.1.5 Cladistics 524 6.2 The five-kingdom system 526 6.3 Protists 528 6.3.1 Protozoan protists 528 6.3.2 Photosynthetic protists 529 6.3.3 Slime mold 530 6.3.4 Oomycetes 531 6.4 Fungi 531 6.4.1 Mycorrhiza 533 6.4.2 Lichens 533 6.5 Plantae 534 6.5.1 Plant life cycle 534 6.5.2 Algae 536 6.5.3 Life cycle of land plants 538 6.5.4 Bryophytes 539 6.5.5 Pteridophytes 541 6.5.6 Gymnosperm 542 6.5.7 Angiosperms 543 6.6 Animalia 547 6.7 Animal’s classification 553 6.7.1 Phylum Porifera 554 6.7.2 Phylum Cnidaria 554 6.7.3 Phylum Platyhelminthes 554 6.7.4 Phylum Aschelminthes 555 6.7.5 Phylum Annelida 557 6.7.6 Phylum Mollusca 557 6.7.7 Phylum Arthropoda 557 6.7.8 Phylum Echinodermata 558 6.7.9 Phylum Hemichordata 558 6.7.10 Phylum Chordata 559 Answers of self test 567 Index xi This is a preview. The total pages displayed will be limited.  Chapter 01 Biomolecules and Catalysis A biomolecule is an organic molecule that is produced by a living organism. Biomolecules act as building blocks of life and perform important functions in living organisms. More than 25 naturally occurring chemical elements are found in biomolecules. Most of the elements have relatively low atomic numbers. Biomolecules consist primarily of carbon, hydrogen, nitrogen, oxygen, phosphorus and sulfur. The four most abundant elements in living organisms, in terms of the percentage of the total number of atoms, are hydrogen, oxygen, nitrogen, and carbon, which together make up over 99% of the mass of most cells. Nearly all of the biomolecules in a cell are carbon compounds, which account for more than one-half of the dry weight of the cells. Covalent bonding between carbon and other elements permit formation of a large number of compounds. Most biomolecules can be regarded as derivatives of hydrocarbons. The hydrogen atoms may be replaced by a variety of functional groups to yield different families of organic compounds. Typical families of organic compounds are the alcohols, which have one or more hydroxyl groups; amines, which have amino groups; aldehydes and ketones, which have carbonyl groups; and carboxylic acids, which have carboxyl groups. Many biomolecules are polyfunctional, containing two or more different kinds of functional groups. Functional groups determine chemical properties of biomolecules. Sugars, fatty acids, amino acids and nucleotides constitute the four major families of biomolecules in cells. Many of the biomolecules found within cells are macromolecules and mostly are polymers (composed of small, covalently linked monomeric subunits). These macromolecules are proteins, carbohydrates, lipids and nucleic acids. Small biomolecules Macromolecules Sugars Polysaccharide Fatty acids Fats/Lipids Amino acids Proteins Nucleotide Nucleic acid Nucleic acids and proteins are informational macromolecules. Proteins are polymers of amino acids and constitute the largest fraction (besides water) of cells. The nucleic acids, DNA and RNA, are polymers of nucleotides. They store, transmit, and translate genetic information. The polysaccharides, polymers of simple sugars, have two major functions. They serve as energy-yielding fuel stores and as extracellular structural elements. 1.1 Amino acids and Proteins Amino acids are compounds containing carbon, hydrogen, oxygen and nitrogen. They serve as monomers (building blocks) of proteins and composed of an amino group, a carboxyl group, a hydrogen atom, and a distinctive side chain, all bonded to a carbon atom, the α-carbon. In an α-amino acid, the amino and carboxylate groups are attached to the same carbon atom, which is called the α-carbon. The various α-amino acids differ with respect to the side chain (R group) attached to their α-carbon. The general structure of an amino acid is: 1 Biomolecules and Catalysis — COO + a H3N C H R (side chain) Figure 1.1 General structure of an amino acid. This structure is common to all except one of the α-amino acids (proline is the exception). The R group or side chain attached to the α-carbon is different in each amino acid. In the simplest case, the R group is a hydrogen atom and amino acid is glycine. — COO e d g b a + 6 5 4 3 2 1 + — H3N C H NH3 CH2 CH2 CH2 CH2 CH COO + H NH3 Figure 1.2 Structure of glycine and lysine. In α-amino acids both the amino group and the carboxyl group are attached to the same carbon atom. However, many naturally occurring amino acids not found in protein, have structures that differ from the α-amino acids. In these compounds the amino group is attached to a carbon atom other than the α-carbon atom and they are called β, γ, δ, or ε amino acids depending upon the location of the C-atom to which amino group is attached. Amino acids can act as acids and bases When an amino acid is dissolved in water, it exists in solution as the dipolar ion or zwitterion. A zwitterion can act as either an acid (proton donor) or a base (proton acceptor). Hence, an amino acid is an amphoteric molecule. At high concentrations of hydrogen ions (low pH), the carboxyl group accepts a proton and becomes uncharged, so that the overall charge on the molecule is positive. Similarly at low concentrations of hydrogen ion (high pH), the amino group loses its proton and becomes uncharged; thus the overall charge on the molecule is negative. R O R O R O + + — — H3N C C OH H3N C C O H2N C C O H H H Low pH (pH < pI) Intermediate pH High pH (pH > pI) (pH = pI) Figure 1.3 The acid-base behavior of an amino acid in solution. At low pH, the positively charged species predominates. As the pH increases, the electrically neutral zwitterion becomes predominant. At higher pH, the negatively charged species predominates. 1.1.1 Optical properties All amino acids except glycine are optically active i.e. they rotate the plane of plane polarized light. Optically active molecules contain chiral carbon. A tetrahedral carbon atom with four different constituents are said to be chiral. All amino acids except glycine have chiral carbon and hence they are optically active. 2 Pages 3 to 33 are not shown in this preview. Biomolecules and Catalysis 1.4 Protein sequencing and assays Determination of amino acid compositions Peptide bonds of proteins are hydrolyzed by either strong acid or strong base. In acid hydrolysis, the peptide can be hydrolyzed into its constituent amino acids by heating it in 6 M HCl at 110°C for 24 hours. Base hydrolysis of polypeptides is carried out in 2 to 4 M NaOH at 100°C for 4 to 8 hours. A mixture of amino acids in hydrolysates can be separated by ion exchange chromatography or by reversed phase HPLC. The identity of the amino acid is revealed by its elution volume and quantified by reaction with ninhydrin. N-terminal analysis Reagent 1-fluoro-2,4-dinitrobenzene (FDNB) and Dansyl chloride are used for determination of N-terminal amino acid residue. FDNB reacts in alkaline solution (pH 9.5) with the free amino group of the N-terminal amino acid residue of a peptide to form a characteristic yellow dinitrophenyl (DNP) derivative. It can be released from the peptide by either acid or enzymic hydrolysis of the peptide bond and subsequently identified. Sanger first used this reaction to determine the primary structure of the polypeptide hormone insulin. This reagent is often referred to as Sanger’s reagent. NO2 NO2 R R O2N F + NH2 C COOH O2N N C COOH + HF H H H FDNB Yellow-coloured derivative Figure 1.34 FDNB reacts with free amino group to produce dinitrophenyl (DNP) derivative of amino acid. Similarly, Dansyl chloride reacts with a free amino group of the N-terminal amino acid residue of a peptide in alkaline solution to form strongly fluorescent derivatives of free amino acids and N-terminal amino acid residue of peptides. Edman degradation Edman degradation method for determining the sequence of peptides and proteins from their N-terminus was developed by Pehr Edman. This chemical method uses phenylisothiocyanate (also termed Edman reagent) for sequential removal of amino acid residues from the N-terminus of a polypeptide chain. Polypeptide R A1 R A2 R A3 R A4 R A5 First round Labeling R A1 R A2 R A3 R A4 R A5 Release Figure 1.35 R A1 R A2 R A3 R A4 R A5 Edman degradation sequentially Labeling removes one residue at a time from Second round the amino end of a peptide. The labeled R A2 R A3 R A4 R A5 amino-terminal residue (R1) can be released without hydrolyzing the rest of the peptide Release bonds. Hence, the amino-terminal residue of the shortened peptide (R2—R3—R4—R5) R A2 R A3 R A4 R A5 can be determined in the second round. 34 This page intentionally left blank. Biomolecules and Catalysis trypsin, chymotrypsin, elastase, thermolysin and pepsin. Various other chemicals also cleave polypeptide chains at specific locations. The most widely used is cyanogen bromide (CNBr), which cleaves peptide bond at C-terminal of Met residues. Similarly hydroxylamine cleaves the polypeptide chain at Asn-Gly sequences. Table 1.8 Specificities of proteolytic enzymes. Rn–1 O Rn O NH CH C NH CH C Agents Site of Cleavage Trypsin Carboxyl side of Lys or Arg, Rn ≠ Pro Chymotrypsin Carboxyl side of aromatic amino acid residues, Rn ≠ Pro Pepsin Amino side of aromatic amino acids like Tyr, Phe and Trp, Rn–1 ≠ Pro Elastase Carboxyl side of Ala, Gly and Ser, Rn ≠ Pro Carboxypeptidases and aminopeptidases are exopeptidases that remove terminal amino acid residues from C and N-termini of polypeptides, respectively. Carboxypeptidase A cleaves the C-terminal peptide bond of all amino acid residues except Pro, Lys and Arg. Carboxypeptidase B is effective only when Arg or Lys are the C-terminal residues. Carboxypeptidase C acts on any C-terminal residue. Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of the protein. Aminopeptidase M catalyzes the cleavage of all free N-terminal residues. Cleavage of disulfide bonds If protein is made up of two or more polypeptide chains and held together by noncovalent bonds then denaturing agents, such as urea or guanidine hydrochloride, are used to dissociate the chains from one another. But polypeptide chains linked by disulfide bonds can be separated by two common methods. These methods are used to break disulfide bonds and also to prevent their reformation. Oxidation of disulfide bonds with performic acid produces two cysteic acid residues. Because these cysteic acid side chains are ionized SO3– groups, electrostatic repulsion prevents S-S recombination. The second method involves the reduction by β-mercaptoethanol or dithiothreitol (Cleland’s reagent) to form cysteine residues. This reaction is followed by further modification of the reactive –SH groups to prevent reformation of the disulfide bond. Acetylation by iodoacetate serves this purpose which modifies the –SH group. Protein assays To determine the amount of protein in an unknown sample is termed as protein assays. The simplest and most direct assay method for proteins in solution is to measure the absorbance at 280 nm (UV range). Amino acids containing aromatic side chains (i.e. tyrosine, tryptophan and phenylalanine) exhibit strong UV-light absorption. Consequently, proteins absorb UV-light in proportion to their aromatic amino acid content and total concentration. Several colorimetric, reagent-based protein assay techniques have also been developed. Protein is added to the reagent, producing a color change in proportion to the amount added. Protein concentration is determined by reference to a standard curve consisting of known concentrations of a purified reference protein. Some most commonly used colorimetric, reagent-based methods are: Biuret method : Biuret method is based on the direct complex formation between the peptide bonds of the protein and Cu2+ ion. This method is not highly sensitive since the complex does not have a high extinction coefficient. Folin method : The Folin assay (also called Lowry method) is dependent on the presence of aromatic amino acids in the protein. First, a cupric/peptide bond complex is formed and then this is enhanced by a phosphomolybodate complex with the aromatic amino acids. Bradford method : Bradford method is based on a blue dye (Coomassie Brilliant Blue) that binds to free amino groups in the side chains of amino acids, especially Lys. This assay is as sensitive as the Folin assay. 36 This page intentionally left blank. Pages 38 to 41 are not shown in this preview. Biomolecules and Catalysis 1.5 Nucleic acids Nucleic acid was first discovered by Friedrich Miescher from the nuclei of the pus cells (Leukocytes) from discarded surgical bandages and called it nuclein. Nuclein was later shown to be a mixture of a basic protein and a phosphorus- containing organic acid, now called nucleic acid. There are two types of nucleic acids (polynucleotides): ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). 1.5.1 Nucleotides The monomeric units of nucleic acids are called nucleotides. Nucleic acids therefore are also called polynucleotides. Nucleotides are phosphate esters of nucleosides and made up of three components: 1. A base that has a nitrogen atom (nitrogenous base) 2. A five carbon sugar 3. An ion of phosphoric acid Nitrogenous bases Nitrogenous bases are heterocyclic, planar and relatively water insoluble aromatic molecules. There are two general types of nitrogenous bases in both DNA and RNA, pyrimidines and purines. H H 7 C6 5 N C4 5 1N C 3N CH 8 2 CH 2 HC C HC CH 4 N9 6 N H N 3 1 Purine Pyrimidine Purines Two different nitrogenous bases with a purine ring (composed of carbon and nitrogen) are found in DNA. The two common purine bases found in DNA and RNA are adenine (6-aminopurine) and guanine (6-oxy-2-aminopurine). Adenine has an amino group (–NH2) on the C6 position of the ring (carbon at position 6 of the ring). Guanine has an amino group at the C2 position and a carbonyl group at the C6 position. Pyrimidines The two major pyrimidine bases found in DNA are thymine (5-methyl-2,4-dioxypyrimidine) and cytosine (2-oxy-4- aminopyrimidine) and in RNA they are uracil (2,4-dioxypyrimidine) and cytosine. Thymine contains a methyl group at the C5 position with carbonyl groups at the C4 and C2 positions. Cytosine contains a hydrogen atom at the C5 position and an amino group at C4. Uracil is similar to thymine but lacks the methyl group at the C5 position. Uracil is not usually found in DNA. It is a component of RNA. NH2 O NH2 O O C C C C C N N N C HN C N CH HN CH HN C CH3 CH CH HC C C C C CH C CH C CH N H2N N O N O N O N N H N H H H H Adenine Guanine Cytosine Uracil Thymine Sugars Naturally occurring nucleic acids have two types of pentose sugars: Ribose and deoxyribose sugar. All known sugars in nucleic acids have the D-stereoisomeric configuration. 42  Biomolecules and Catalysis Ribose sugar is found in RNA. β-D-Ribose is a five carbon sugar with a hydroxyl group (–OH) on each carbon (the carbon atoms of the ribose/deoxyribose present in nucleoside/nucleotides are designated with a prime (’) mark to distinguish them from the backbone numbering in the bases). Deoxyribose sugar is found in DNA. The hydroxyl group at 2’ position of ribose sugar is replaced by a hydrogen (–H). 5’ 5’ HOCH2 OH HOCH2 OH O O 4’ 1’ 4’ 1’ H 3’ 2’ H H 3’ 2’ H HO OH HO H b-D-Ribose b-D-2-Deoxyribose Sugar pucker Pentose sugar is non-planar. This non-planarity is termed puckering. Pentose ring can be puckered in two basic conformations: envelope and twisted. In the envelope form, the four carbons of the pentose sugar are nearly coplanar and the fifth is away from the plane. In twisted form three atoms are coplanar and the other two lie away on opposite sides of this plane. Twisting the C2’ and C3’ carbons relative to the other atoms results in twisted forms of the sugar ring. Sugar pucker can be endo or exo. C2’ or C3’ endo pucker means that C2’ or C3’ are on the same side as the base and C4’-C5’ bond. Exo-pucker describes a shift in the opposite direction. Purines show a preference for the C2’- endo pucker conformational type whereas pyrimidines favour C3’- endo. In RNA we find predominantly the C3’-endo conformation. 5’ C N 5’ C 3’ N 3’ O 1’ 4’ 4’ O 1’ 2’ 2’ Envelope form, C3' endo Twisted form, C3' endo and C2' exo 5’ C 5’ C N N 2’ 2’ O 1’ O 4’ 4’ 1’ 3’ 3’ Envelope form, C2' endo Twisted form, C2' endo and C3' exo Figure 1.38 Sugar puckers. Nucleoside Sugar and nitrogenous base join to form nucleoside. The bond between the sugar and the base is called the glycosidic bond. This bond is said to be in the β (up) configuration with respect to the ribose sugar. 9 1 5’ 5’ HOCH2 b-glycosidic HOCH2 b-glycosidic O bond O bond 4’ 1’ 4’ 1’ H H H H 3’ 2’ H 3’ 2’ H HO OH HO OH Figure 1.39 Structure of nucleoside. 43 This page intentionally left blank.  Biomolecules and Catalysis Table 1.9 Naming nucleosides and nucleotides Bases Purines Pyrimidines Adenine (A) Guanine (G) Cytosine (C) Uracil (U)/Thymine (T) Nucleosides–in RNA Adenosine Guanosine Cytidine Uridine in DNA Deoxyadenosine Deoxyguanosine Deoxycytidine Deoxythymidine Nucleotides– in RNA Adenylate Guanylate Cytidylate Uridylate in DNA Deoxyadenylate Deoxyguanylate Deoxycytidylate Deoxythymidylate Nucleoside monophosphate AMP GMP CMP UMP/TMP Nucleoside diphosphate ADP GDP CDP UDP/TDP Nucleoside triphosphate ATP GTP CTP UTP/TTP Polynucleotides Polynucleotides are formed by the condensation of two or more nucleotides. The condensation most commonly occurs between the alcohol of a 5'-phosphate of one nucleotide and the 3'-hydroxyl of a second, with the elimination of H2O, forming a phosphodiester bond. All nucleotides in a polynucleotide chain have the same relative orientation. The formation of phosphodiester bonds in DNA and RNA exhibits directionality. The primary structure of DNA and RNA (the linear arrangement of the nucleotides) proceeds in the 5'→ →3' direction. The common representation of the primary structure of DNA or RNA molecules is to write the nucleotide sequences from left to right synonymous with the 5'→ → 3' direction as shown below. 5'-pGpApTpC-3' — O Nitrogen base 5' end — O P O CH2 O O H H { H H O H or OH — O P O Phosphodiester bond O Nitrogen base CH2 O H H H H O H or OH — O P O O Nitrogen base CH2 O H H H H OH H or OH 3' end Figure 1.42 The polynucleotide has a 5' end, which is usually attached to a phosphate, and a 3' end, which is usually a free hydroxyl group. The backbones of these polynucleotide are formed by 3' to 5' phosphodiester linkages. 45 Pages 46 to 48 are not shown in this preview.  Biomolecules and Catalysis 1.6.2 Z-DNA Left-handed Z-DNA has been mostly found in alternating purine-pyrimidine sequences (CG)n and (TG)n. Z-DNA is thinner (18 Å) than B-DNA (20 Å), the bases are shifted to the periphery of the helix, and there is only one deep, narrow groove equivalent to the minor groove in B-DNA. In contrast to B-DNA where a repeating unit is a 1 base pair, in Z-DNA the repeating unit is a 2 base pair. The backbone follows a zigzag path as opposed to a smooth path in B-DNA. The sugar and glycosidic bond conformations alternate; C2’ endo in anti dC and C3’ endo in syn dG. Electrostatic interactions play a crucial role in the Z-DNA formation. Therefore, Z-DNA is stabilized by high salt concentrations or polyvalent cations that shield interphosphate repulsion better than monovalent cations. Z-DNA can form in regions of alternating purine-pyrimidine sequence; (GC)n sequences form Z-DNA most easily. (GT)n sequences also form Z-DNA but they require a greater stabilization energy. (AT)n sequences generally does not form Z-DNA since it easily forms cruciforms. Table 1.10 Comparisons of different forms of DNA Geometry attribute A-form B-form Z-form Helix sense Right-handed Right-handed Left-handed Repeating unit 1 bp 1 bp 2 bp Rotation/bp (Twist angle) 33.6° 34.3° 60°/2 Mean bp/turn 10.7 10.4 12 Base pair tilt 20° –6° 7° Rise/bp along axis 2.3Å 3.32Å 3.8Å Pitch/turn of helix 24.6Å 33.2Å 45.6Å Mean propeller twist +18° +16° 0° Glycosidic bond conformation Anti Anti Anti for C, Syn for G Sugar pucker C3'-endo C2'-endo C:C2'-endo, G:C3'-endo Diameter 23Å 20Å 18Å Major groove Narrow and deep Wide and deep Flat Minor groove Wide and shallow Narrow and deep Narrow and deep 1.6.3 Triplex DNA In certain circumstances (e.g., low pH), a DNA sequence containing a long segment consisting of a polypurine strand, hydrogen bonded to a polypyrimidine strand and form a triple helix. The triple helix will be written as (dT).(dA).(dT) with the third strand in italics. Triple-stranded DNA is formed by laying a third strand into the major groove of DNA. A third strand makes a hydrogen bond to another surface of the duplex. The third strand pairs in a Hoogsteen base-pairing scheme. The central strand of the triplex must be purine rich. Thus, triple-stranded DNA requires a homopurine: homopyrimidine region of DNA. If the third strand is purine rich, it forms reverse Hoogsteen hydrogen bonds in an antiparallel orientation with the purine strand of the Watson-Crick helix. If the third strand is pyrimidine rich, it forms Hoogsteen bonds in a parallel orientation with the Watson-Crick-paired purine strand. Triple helix can be intermolecular or intramolecular. In the intermolecular Pu.Pu.Py triple helix, the poly-purine third strand is organized antiparallel with respect to the purine strand of the original Watson-Crick duplex. In the intermolecular Py.Pu.Py triplex, the polypyrimidine third strand is organized parallel with respect to the purine strand and the phosphate backbone is positioned. 49 Biomolecules and Catalysis 5' 3’ Polypyrimidine strand Polypurine third strand Polypurine strand Figure 1.45 Intermolecular Pu.Pu.Py triple 5' helix. The polypurine third strand (black colour) is organized antiparallel with respect to the purine strand of 5' the original double strand DNA. 3’ An intramolecular triplex (also referred to as H-DNA) could form within a single homopurine.homopyrimidine duplex DNA region in the supercoiled DNA. As in intermolecular triplexes, when the third strand is the pyrimidine strand, it forms Hoogsteen pairs in a parallel fashion with the central purine strand. When the third strand is the purine strand, it forms reverse Hoogsteen pairs in an antiparallel fashion with the central purine strand. 1.6.4 G-quadruplex Nucleic acid sequences which are rich in guanine are capable of forming four-stranded structures called G-quadruplexes (also called G-quartat). These consist of a square arrangement of guanines (a tetrad), stabilized by Hoogsteen hydrogen bonding. The formation and stability of the G-quadruplexes is a monovalent cation-dependent. A monovalent cation is presents in the center of the tetrads. G-quadruplexes can be formed of DNA or RNA. They can be formed from one, two or four separate strands of DNA or RNA. Depending on the direction of the strands or parts of a strand that form the tetrads, structures may be described as parallel or antiparallel. All parallel quadruplexes have all guanine glycosidic angles in an anti conformation. Anti-parallel quadruplexes have both syn and anti conformations. H Anti N N N H N N Anti N H N O N O N H + N H H M H H N O H N O N H N N N Figure 1.46 N N N H N Four-stranded structures can arise Anti H Anti from square arrangement of guanines. 50 Pages 51 to 57 are not shown in this preview. Biomolecules and Catalysis 1.7 RNA DNA contains all the information needed to maintain a cell’s processes, but these precious blueprints never leave the protected nucleus. How, then, all these data are transmitted to the body of the cell itself where they are put to use? The answer: by way of RNA. RNA molecules play essential roles in the transfer of genetic information during protein synthesis and in the control of gene expression. The diverse functions of RNA molecules in living organisms also include the enzymatic activity of ribozymes and the storage of genetic information in RNA viruses and viroids. So, RNA may be genetic or non genetic, catalytic or non-catalytic and coding (mRNA) or noncoding (like tRNA, rRNA). Thermodynamic stability of RNA structure Primary structure of RNA refers to the sequence of nucleotides. Secondary structure in RNA is dominated by Watson-Crick base pairing. This fundamental interaction between bases leads to the formation of double-helical structures of varying length. In RNA, double-helical tracts are generally short. RNA double helices adopt the A-form structure, which differs significantly from the canonical B-form adopted by DNA double helices. RNA’s secondary structure is generally more stable than its tertiary structure. Thus, formation of the secondary structure dominates the process of RNA folding. RNA tertiary structure forms through relatively weak interactions between preformed secondary structure elements. RNA duplexes are more stable than DNA duplexes. At physiological pH, denaturation of a double stranded helical RNA often requires higher temperatures than those required for denaturation of a DNA molecule with a comparable sequence. However, the physical basis for these differences in thermal stability is not known. Types of RNA Within a given cell, RNA molecules are found in multiple copies and in multiple forms. Major RNA classes are mRNA, rRNA, tRNA, snRNA, SnoRNA, miRNA, XIST, scRNA, siRNA, tmRNA and telomerase RNA. Features of few major forms of RNA present in prokaryotic and eukaryotic cells are given below. mRNA mRNA (messenger RNA) carries the genetic information copied from DNA in the form of a series of three-base code words, each of which specifies a particular amino acid. Most of the eukaryotic mRNAs represent only a single gene: they are monocistronic. mRNAs, which carry sequence coding for several polypeptides are called polycistronic. In these cases, a single mRNA is transcribed from a group of adjacent genes. Most of the prokaryotic mRNA are polycistronic. All mRNAs contain two types of regions. The coding region consists of a series of codons starting with an AUG and ending with a termination codon. But the mRNA is always longer than the coding region, extra regions are present at both ends. The untranslated region at the 5’ end is described as the leader and untranslated region at the 3’ end is called the trailer. A polycistronic mRNA also contains intercistronic regions. They vary greatly in size. They may be as long as 30 nucleotides. Eukaryotic mRNA molecules often require extensive processing and transport, while prokaryotic molecules do not. tRNA tRNA is a small, well-characterized RNA molecules with a key role in protein biosynthesis. Transfer RNA is also known as adaptor RNA. The concept of an adaptor to provide the interface between nucleic acid language and protein language was introduced by Crick in 1955. tRNAs also participate in nonprotein synthetic processes such as a primer during reverse transcription in retrovirus life cycles. tRNA is a single RNA chain of 73-93 nucleotides, present in the cytosol and organelles of all living cells. Holley and his co-workers determined the first tRNA sequence in 1965. Dictated by their primary sequence, tRNA folds into cloverleaf-like secondary structures with well-defined stems and loops that make up the acceptor arm, D arm and loop, anticodon arm and loop, and the T-arm and loop. Regardless of the length of the tRNA, the numbering of conserved nucleotides remains constant. 58 This page intentionally left blank. Biomolecules and Catalysis maintaining the telomeres. They are always associated with specific proteins, and the complexes are referred to as small nuclear ribonucleoproteins (snRNP) or sometimes as snurps. snoRNA snoRNA (small nucleolar RNA) is a class of small RNA molecules that are involved in chemical modifications such as methylation of rRNAs and other forms of RNA in eukaryotes. snoRNAs form a component in the small nucleolar ribonucleoprotein (snoRNP), which contains snoRNA and proteins. The snoRNA guides the snoRNP complex to the modification site of the target RNA gene via sequences in the snoRNA that hybridize to the target site. The proteins then catalyze modification of the RNA gene. gRNAs gRNAs (guide RNA) are RNA genes that function in RNA editing. RNA editing was first reported in the mitochondria of kinetoplastids, in which mRNAs are edited by inserting or deleting stretches of uridylates (Us). The gRNA forms part of the editosome and contains sequences that hybridize to matching sequences in the mRNA, to guide the mRNA modifications. Small silencing RNAs Small silencing RNAs are small RNA of ~20–30 nucleotides long and make an association with members of the Argonaute protein family, which they guide to their regulatory targets. After the discovery of the first small silencing RNA in year 1993, several small RNA classes have been identified which differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. The most common examples of these small RNAs are siRNA (small interfering RNA), miRNA (micro RNA) and piRNA (Piwi-interacting RNA). miRNAs are endogenous regulatory RNAs which are typically 20–25 nucleotides long, and are thought to regulate the expression of other genes. miRNAs derive from precursor transcripts called primary miRNAs (pri-miRNAs), which are typically transcribed by RNA polymerase II. The pri-miRNA is processed in the nucleus into a 60-70 nucleotide pre-miRNA by the activity of Drosha, a nuclear enzyme. The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by exportin protein. The Dicer enzyme, a dsRNA specific RNaseIII family endonuclease, then cuts pre-miRNA into the mature miRNA. siRNAs are small RNA molecules of ~21 nucleotides. siRNA duplexes are produced by the Dicer. Out of two strands, the one that directs silencing of target mRNA is called guide RNA. Whereas the other strand which is ultimately destroyed, is the passenger strand. Target regulation is mediated by RISC (RNA Induced Silencing Complex). siRNA may be exo-siRNA and endo siRNA depending on the source of RNA. tmRNA tmRNA has a complex structure with tRNA-like and mRNA-like regions. It has currently only been found in bacteria. tmRNA recognizes ribosomes that have trouble translating or reading an mRNA and stall, leaving an unfinished protein that may be detrimental to the cell. tmRNA acts like a tRNA first, and then an mRNA that encodes a peptide tag. The ribosome translates this mRNA region of tmRNA and attaches the encoded peptide tag to the C-terminus of the unfinished protein. This attached tag targets the protein for destruction or proteolysis. 1.7.1 Alkali-catalyzed cleavage of RNA Under alkaline conditions, RNA is hydrolyzed rapidly and generates a mixture of 2’- and 3’ nucleoside monophosphate. In the presence of a hydroxide ion, the 2’-hydroxyl group of the ribose is converted to a 2’-alkoxide ion. The 2’-alkoxide attacks the 3’-phosphodiester group, breaking the 5’-3’ phosphodiester bond and forming a cyclic 2’,3’-nucleoside monophosphate. Another hydroxide ion attacks the cyclic 2’,3’-nucleoside monophosphate, yielding a mixture of 2’-and 3’-nucleoside monophosphates. DNA is stable in basic solution because DNA lacks a 2’-hydroxyl group to carry out intramolecule catalysis. 60  Biomolecules and Catalysis A CH2 O H H H H 2’-Nucleoside OH O monophosphate Cyclic 2’,3’- nucleoside monophosphate — O P O A O — CH2 O A Hydrolysis CH2 O or H H H H H H A O O H H H CH2 — P O O O O — H O O H H — O P O Transesterification + H H 3’-Nucleoside O HO O OH monophosphate T T — CH2 O CH2 O O P O — H H H H O H H H H O OH O OH Figure 1.55 Alkali-catalyzed cleavage of RNA. 1.7.2 RNA world hypothesis The RNA world hypothesis proposes that RNA was actually the first life-form on earth, later developing a cell membrane around it and becoming the first prokaryotic cell (the phrase RNA World was first used by Walter Gilbert in 1986). This hypothesis is supported by the RNA’s ability to store, transmit, and duplicate genetic information, just like DNA does and to catalyze chemical reactions, just like protein does. RNA with catalytic activity is termed as ribozyme. Because RNA can perform the tasks of both genetic materials and enzymes, RNA is believed to have once been capable of independent life. 1.7.3 RNA as genetic material Some viruses contain an RNA as genetic material. One of the first experiments that established RNA as the genetic material in RNA viruses was the reconstitution experiment of H.Fraenkel-Conrat and B.Singer. Degraded Protein A TMV type A RNA A Infection of tobacco leaf Degraded TMV type B RNA B TMV type B RNA B Figure 1.56 Proof that the genetic material of tobacco mosaic virus (TMV) is RNA, not protein. 61 This page intentionally left blank.  Biomolecules and Catalysis 1.8 Carbohydrates Carbohydrates are polyhydroxy aldehydes or polyhydroxy ketones, or compounds that can be hydrolyzed to them. In the majority of carbohydrates, H and O are present in the same ratio as in water, hence also called as hydrates of carbon. Carbohydrates are the most abundant biomolecules on Earth. Carbohydrates are classified into following classes depending upon whether these undergo hydrolysis and if so on the number of products form: Monosaccharides are simple carbohydrates that cannot be hydrolyzed further into polyhydroxy aldehyde or ketone unit. Oligosaccharides are polymers made up of two to ten monosaccharide units joined together by glycosidic linkages. Oligosaccharides can be classified as di-, tri-, tetra- depending upon the number of monosaccharides present. Amongst these the most abundant are the disaccharides, with two monosaccharide units. Polysaccharides are polymers with hundreds or thousands of monosaccharide units. Polysaccharides are not sweet in taste hence they are also called non-sugars. 1.8.1 Monosaccharide Monosaccharides consist of a single polyhydroxy aldehyde or ketone unit. Monosaccharides are the simple sugars, which cannot be hydrolyzed further into simpler forms and they have a general formula CnH2nOn. Monosaccharides are colourless, crystalline solids that are freely soluble in water but insoluble in nonpolar solvents. The most abundant monosaccharide in nature is the D-glucose. Monosaccharides can be further sub classified on the basis of: The number of the carbon atoms present Monosaccharides can be named by a system that is based on the number of carbons with the suffix-ose added. Monosaccharides with four, five, six and seven carbon atoms are called tetroses, pentoses, hexoses and heptoses, respectively. System for numbering the carbons : The carbons are numbered sequentially with the aldehyde or ketone group being on the carbon with the lowest possible number. 1 CHO 6 CHO H C OH H C OH 2 5 HO C H HO C H 3 4 H C OH H C OH 4 3 H C OH H C OH 5 2 6 CH2OH 1 CH2OH Correct Incorrect Presence of aldehydes or ketones groups Aldoses are monosaccharides with an aldehyde group. Ketoses are monosaccharides containing a ketone group. The monosaccharide glucose is an aldohexose; that is, it is a six-carbon monosaccharide (-hexose) containing an aldehyde group (aldo-). Similarly fructose is a ketohexose; that is, it is a six-carbon monosaccharide (-hexose) and containing a ketone group (keto-). Trioses are simplest monosaccharides. There are two trioses– dihydroxyacetone and glyceraldehyde. Dihydroxyacetone is called a ketose because it contains a keto group, whereas glyceraldehyde is called an aldose because it contains an aldehyde group. 63 Biomolecules and Catalysis H H C1 O H C1 OH H C2 OH C2 O H C3 OH H C3 OH H H Glyceraldehyde Dihydroxyacetone (an aldose) (a ketose) Figure 1.57 Trioses, the simplest monosaccharides. Glyceraldehyde has a central carbon (C–2) which is chiral or asymmetrical. Chiral molecules such as glyceraldehyde can exist in two forms or configurations that are non-superimposable mirror images of each other. These two forms are called enantiomers. An enantiomer is identified by its absolute configuration. Glyceraldehyde has two absolute configurations. When the hydroxyl group attached to the chiral carbon is on the left in a Fischer projection, the configuration is L; when the hydroxyl group is on the right, the configuration is D. The absolute configurations of monosaccharide containing more than one chiral centers like hexose are determined by comparing the configuration at the highest-numbered chiral carbon (the chiral carbon farthest from the aldehyde group) to the configuration at the single chiral carbon of glyceraldehyde. 1 CHO 1 CHO H C OH HO C H 2 2 H C OH H C OH 3 3 H H D-Glyceraldehyde L-Glyceraldehyde Figure 1.58 The enantiomers of glyceraldehyde. The configuration of groups around the chiral carbon 2 (shown in bold) distinguishes D-glyceraldehyde from L-glyceraldehyde. The two molecules are mirror images and cannot be superimposed on one another. All the monosaccharides except dihydroxyacetone contain one or more chiral carbon atoms and thus occur in optically active isomeric forms. As the number of chiral carbon atoms increases, the number of possible stereoisomers also increases. The total number of possible isomers can be determined by using Van’t Hoff’s rule. A compound with ‘n’ chiral carbon atom has a maximum of 2n possible stereoisomers. 1.8.2 Epimers Many common sugars are closely related, differing only by the stereochemistry at a single carbon atom. For example, D-glucose and D-mannose differ only at carbon 2. Sugars that differ only by the stereochemistry at a single carbon (other than anomeric carbon) are called epimers. Similarly D-glucose and D-galactose are epimers. D-mannose and D-galactose are not epimers because their configuration differ at more than one carbon. 64 Pages 65 to 72 are not shown in this preview.  Biomolecules and Catalysis O-linked glycosidic bond N-linked glycosidic bond CH2OH CH2OH C O O C O O O CH2 CH Ser O NH C CH2 CH Asn 1 1 OH NH OH NH O H O H Monosaccharide Monosaccharide Core protein Core protein Figure 1.64 Carbohydrate groups are covalently attached to many different proteins to form glycoproteins. Sugars are attached either to the amide nitrogen atom in the side chain of asparagine (termed an N-linkage) or to the oxygen atom in the side chain of serine or threonine (termed an O-linkage). 1.8.8 Reducing and non-reducing sugar Sugars capable of reducing ferric or cupric ion are called reducing sugar. A reducing sugar is any sugar that either has an aldehyde group or is capable of forming one in solution through isomerisation. This functional group allows the sugar to act as a reducing agent. All monosaccharides whether aldoses and ketoses, in their hemiacetal and hemiketal form are reducing sugars. All disaccharides formed from head to tail condensation are also reducing sugar i.e. disaccharides except sucrose, trehalose are reducing sugars. All reducing sugars undergo mutarotation in aqueous solution. Sugars like sucrose, trehalose not capable of reducing ferric or cupric ion are called non-reducing sugar. In sucrose and trehalose, anomeric carbon becomes involved in a glycosidic bond. So they donot contain free anomeric carbon atoms. Sucrose and trehalose are therefore not a reducing sugar, and have no reducing end. So it cannot be oxidized by cupric or ferric ion. In describing disaccharides or polysaccharides, the end of a chain that has a free anomeric carbon (i.e. is not involved in a glycosidic bond) is commonly called the reducing end of the chain. 1.9 Lipids Biological lipids are a chemically diverse group of organic compounds which are insoluble or only poorly soluble in water. They are readily soluble in nonpolar solvents such as ether, chloroform, or benzene. The hydrophobic nature of lipids is due to the predominance of hydrocarbon chains (—CH2—CH2—CH2—) in their structures. Unlike the proteins, nucleic acids, and polysaccharides, lipids are not polymers. Functions Biological lipids have diverse functions. The four general functions of biological lipids have been identified. They serve as a storage form of metabolic fuel. They serve as a transport form of metabolic fuel. They provide the structural components of membranes. They have protective functions in bacteria, plants, insects, and vertebrates, serving as a part of the outer coating between the body of the organism and the environment. Apart from the general functions biological lipids serve as pigments (carotene), hormones (vitamin D derivatives, sex hormones), signaling molecules (eicosanoids, phosphatidylinositol derivatives), cofactors (vitamin K), detergents (bile salt) and many other specialized functions. 73 This page intentionally left blank.  Biomolecules and Catalysis The notation 18:1 denotes a 18 carbons fatty acid with one double bond, whereas 18:2 signifies that there are two double bonds. The most commonly used systems for designating the position of double bonds in an unsaturated fatty acid is the delta (Δ) numbering system. For example, cis-Δ9 means that there is a cis double bond between carbon atoms 9 and 10; trans-Δ2 means that there is a trans double bond between carbon atoms 2 and 3. In this nomenclature the carboxyl carbon is designated carbon 1. For example, palmitoleic acid has 16 carbons and has a double bond between carbons 9 and 10. It is designated as 16:1:Δ9. There is an alternative convention for naming polyunsaturated fatty acids. In this convention, number 1 is assigned to the methyl carbon. This carbon is also designated ω. The positions of the double bonds are indicated relative to the ω carbon. Essential fatty acids Mammals lack the enzymes to introduce double bonds at carbon atoms beyond C-9 in the fatty acid chain. Hence, mammals cannot synthesize linoleate and linolenate. Linoleate and linolenate are the two essential fatty acids. The term essential means that they must be obtained from the diet because they are required by an organism and cannot be endogenously synthesized. Fatty acids that can be endogenously synthesized are termed as nonessen- tial. They are nonessential also in the sense that they do not have to be obligatorily included in the diet. Melting point of fatty acids The melting point of fatty acids depend on chain length and degree of unsaturation. The longer the chain length, the higher the melting point; and the greater the number of double bonds, the lower the melting point. The presence of double bonds makes unsaturated chain more rigid. As a result, unsaturated chains cannot pack themselves in crystals efficiently and densely as saturated chain, so, they have lower melting point as compared to saturated fatty acids. Similarly, the unsaturated fatty acids with cis configuration have lower melting points than the unsaturated fatty acids with trans configuration. Problem Why unsaturated fatty acids have low melting points? Solution The presence of double bonds makes unsaturated chain more rigid. As a result, unsaturated chains cannot pack themselves in crystals efficiently and densely as saturated chain, so, they have lower melting point as compared to saturated fatty acids. 1.9.2 Triacylglycerol and Wax Triacylglycerols (also called triglycerides) are triesters of fatty acids and glycerol. They are composed of three fatty acids and a glycerol molecule. Triacylglycerols are of two types – simple and mixed type. Those containing a single kind of fatty acids are called simple triacylglycerols and with two or more different kinds of fatty acids are called mixed triacylglycerols. The general formula of triacylglycerol is given below: O O 16 H2C O C H2C O C R1 O O 18 H C O C H C O C R2 O O 18 H2C O C H2C O C R3 Figure 1.66 General structure of triacylglycerol. 75 Biomolecules and Catalysis Triacylglycerols are nonpolar, hydrophobic in nature and a major form of stored lipids. Because triacylglycerols have no charge (i.e. the carboxyl group of each fatty acid is joined to glycerol through a covalent bond), they are sometimes referred to as neutral lipid. Triacylglycerol molecules contain fatty acids of varying lengths, which may be unsaturated or saturated. Triacylglycerols can be distinguished as fat and oil on the basis of physical state at room temperature. Fats, which are solid at room temperature, contain a large proportion of saturated fatty acids. Oils are liquid at room temperature because of their relatively high unsaturated fatty acid content. Hydrolysis of triacylglycerols with alkalis such as NaOH or KOH is called saponification. Saponification yields salts of free fatty acids (termed soap) and glycerol. The number of milligrams of KOH required to saponify one-gram of fat is known as saponification number. The saponification number measures the average molecular weight of fats. Similarly, the number of grams of iodine that can be added to 100g sample of fat or oil is called iodine number, which is used to determine the degree of unsaturation (i.e. extent of unsaturation). Waxes Natural waxes are typically esters of fatty acids and long chain alcohols. They are formed by esterification of long chain fatty acids (saturated and unsaturated) and high molecular weight monohydroxy alcohols (C14 to C36). Waxes are biosynthesized by many plants or animals. The best known animal wax is beeswax. Triacontanoylpalmitate (an ester of palmitic acid with the alcohol triacontanol) is the major component of beeswax. O CH3(CH2)n C O CH2(CH2)mCH3 O CH3(CH2)14 C O CH2 (CH2)28 CH3 Palmitic acid 1-triacontanol Figure 1.67 The general structure of a wax. 1.9.3 Phospholipids A phospholipid is an amphipathic molecule constructed from four components: fatty acids, a platform to which the fatty acids are attached, a phosphate and an alcohol attached to the phosphate. The platform on which phospholipids are built may be glycerol or sphingosine. Phosphoglycerides Phospholipids derived from glycerol are called phosphoglycerides (or glycerophospholipids). A phosphoglyceride consists of a glycerol molecule, two fatty acids, a phosphate, and an alcohol (e.g. choline). Phosphoglycerides are the most numerous phospholipid molecules found in cell membranes. In phosphoglycerides, the hydroxyl groups at C-1 and C-2 of glycerol are esterified to the carboxyl groups of the two fatty acid chains. The C-3 hydroxyl group of the glycerol backbone is esterified to phosphoric acid. When no further additions are made, the resulting compound is phosphatidic acid, the simplest phosphoglyceride. Phos- phatidic acids are found in small amount in most natural systems. The major phosphoglycerides are derived from phosphatidic acid by the formation of an ester bond between the phosphate group and the hydroxyl group of one of several alcohols. The common alcohol moieties of phosphoglycerides are serine, ethanolamine, choline, glycerol, and the inositol. If the alcohol is choline, the phosphoglyceride molecule is called phosphatidylcholine (also referred to as lecithin) and if serine then it is called phosphotidylserine. 76 Pages 77 to 81 are not shown in this preview. Biomolecules and Catalysis 1.10 Vitamins Vitamins are organic compounds required by the body in trace amounts to perform specific cellular functions. They can be classified according to their solubility and their functions in metabolism. The requirement for any given vitamin depends on the organisms. Not all vitamins are required by all organisms. Vitamins are not synthesized by humans, and therefore must be supplied by the diet. Vitamins may be water soluble or fat soluble. Nine vitamins (thiamines, riboflavin, niacin, biotin, pantothenic acid, folic acid, cobalamin, pyridoxine, and ascorbic acid) are classified as water soluble, whereas four vitamins (vitamins A, D, E and K) are termed fat-soluble. Except for vitamin C, the water soluble vitamins are all precursors of coenzymes. 1.10.1 Water-soluble vitamins Thiamine (vitamin B1) Thiamine pyrophosphate (TPP) is the biologically active form of the vitamin, formed by the transfer of a pyrophosphate group from ATP to thiamine. Thiamine is composed of a substituted thiazole ring joined to a substituted pyrimidine by a methylene bridge. Thiazolium Aminopyrimidine Reactive H NH2 H NH2 carbon + S N N + S N N AMP ATP N CH3 CH3 N CH3 CH3 O TPP synthetase O — O P O H Thiamine O — O P O — O Thiamine pyrophosphate (TPP) Figure 1.74 Structure of thiamine and thiamine pyrophosphate. TPP serves as a coenzyme in the oxidative decarboxylation of α-keto acid, and in the formation or degradation of α-ketols (hydroxy ketones) by transketolase. Pyruvate decarboxylase Pyruvate (a-keto acid) Acetaldehyde + CO2 Transketolase Xylulose-5-Phosphate + Ribose-5-Phosphate Glyceraldehyde–3–Phosphate + Sedoheptulose–7-Phosphate Beri-Beri is a severe thiamine-deficiency syndrome found in areas where polished rice is the major component of the diet. Riboflavin (vitamin B2) Riboflavin is a constituent of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). FMN is synthesized after the addition of phosphate in riboflavin and FAD formed by the transfer of an AMP moiety from ATP to FMN. FMN and FAD are each capable of reversibly accepting two hydrogen atoms, forming FMNH2 or FADH2. The oxidized form of the isoalloxazine structure absorbs light around 450 nm. The colour is lost, when the ring is reduced. 82  Biomolecules and Catalysis H O O Isoalloxazine H3C N H3C N N NH NH 2H + — N N O N N O 2e N H3C H3C CH2 CH2 H H C OH ADP PPi H C OH FADH2 (Reduced) ATP ATP Ribitol H C OH FMN H C OH H C OH H C OH CH2OH H2C O P P Adenosine Riboflavin FAD (Oxidized) Figure 1.75 Structure and biosynthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Niacin Niacin, or nicotinic acid, is a substituted pyridine derivative. The biologically active coenzyme forms are nicotinamide adenine dinucleotide (NAD+) and its phosphorylated derivative, nicotinamide adenine dinucleotide phosphate (NADP+). Nicotinamide, is a derivative of nicotinic acid that contains an amide instead of a carboxyl group. NAD+ and NADP+ serve as coenzymes in oxidation-reduction reactions in which the coenzyme undergoes reduction of the pyridine ring by accepting a hydride ion (H–). The reduced forms of NAD+ and NADP+ are NADH and NADPH, respectively. O O H H H NH2 C