Міністерство охорони здоров’я України Харківський національний медичний університет Л.Д. Попова, Г.В. Полікарпова БІОХІМІЯ Навчально-методичний посібник для студентів вищих навчальних закладів IV рівня акредитації та лікарів-інтернів L.D. Popova, A.V. Polikarpova BIOCHEMISTRY Manual for medical students and interns Рекомендовано Міністерством освіти і науки України як навчальний посібник для студентів вищих навчальних закладів Х а р к і в 2011 УДК 577.1(075.8)=111 ББК 28.072.я7 B 60 Рекомендовано Міністерством освіти і науки України (08.02.2011) Рецензенти: Є.Е. Перський д-р біол. наук, проф. (Харківський національний університет ім. В.Н. Каразіна); О.І. Залюбовська д-р мед. наук, проф. (Харківський національний фармацевтичний університет); В.В. Давидов д-р мед. наук, проф. (Інститут охорони здоров’я дітей та підлітків АМН України). Reviewers: E.E. Perskiy doctor of biology, professor (Karazin’s Kharkiv national univer- sity); O.I. Zalubovska doctor of medicine, professor (Kharkiv national pharma- ceutical university); V.V. Davidov doctor of medicine, professor (Institute of children’s and uvenil’s health pretection). Автори: Попова Людмила Дмитрівна Полікарпова Ганна Валеріївна Autors: Popova Liudmyla D. Polikarpova Anna V. В 60 Біохімія: Навч.-метод. посібник для студентів та лікарів-інтернів/ Л.Д. Попова, Г.В. Полiкарпова – Харків: ХНМУ, 2011. – 539 с. ISBN B 60 Biochemistry: Manual for medical students or interns/ L.D. Popova, A.V. Polikarpova – Kharkiv: KNMU, 2011. – 539 p. ISBN The manual summarizes current knowledge relevant to the static, dynamic, and functional biochemistry. Structure and metabolism of basic classes of biomolecules (proteins, amino acids, nucleic acid, nucleotides, carbohydrates, lipids) are considered. Modern conceptions of molecular biology and genetics, biochemical bases of physiological functions and neurohumoral regulation are elucidated. Biochemistry of blood, kidney, muscle, liver, immune, nervous, connective tissue is presented. Molecular mechanisms of metabolic disorders of different classes of substances and pathways of their correction are discussed. The book is dedicated to the medical students and interns. УДК 577.1(075.8)=111 ББК 28.072.я7 ISBN © Харківський національний медичний університет, 2011 2 CONTENTSCONTENTS List of Abbreviations 8 Foreword 11 Chapter 1 Structure of Proteins and Amino Acids 12 1.1 Functions of Proteins 12 1.2 Proteinogenic Amino Acids 12 1.3 Levels of Protein Structure 19 1.4 Protein Classification 24 Chapter 2 Carbohydrates and Their Derivatives 28 2.1 Monosaccharides and Their Derivatives 28 2.2 Oligosaccharides 34 2.3 Polysaccharides 35 2.4 Functions of Carbohydrates 40 Chapter 3 Structure and Functions of Lipids 43 3.1 Functions of Lipids 43 3.2 Common Characteristic of Lipids. Fatty Acids 44 3.3 Simple Lipids 46 3.4 Complex Lipids 47 Chapter 4 Structure of Nucleotides and Nucleic acids 54 4.1 Functions of Nucleic Acids and Nucleotides 54 4.2 Structure of Nucleotides 54 4.3 Structure of Nucleic Acids 57 Chapter 5 Enzymes 68 5.1 Structure and Role 68 5.2 Mechanism of Enzyme Action 72 5.3 Kinetics of Enzymatic Catalysis 73 5.4 Regulation of Enzymatic Processes 79 5.5 Classification and Nomenclature of Enzymes 82 5.6 Medical Enzymology 83 Chapter 6 Vitamins 88 6.1 General Characteristic. Classifications. Antivitamins 88 6.2 Fat-soluble Vitamins 94 6.3 Water-soluble Vitamins 102 6.4 Vitamin-like Substances (Vitaminoids) 116 Chapter 7 Principles of Bioenergetics. Biological oxidation. The Citric Acid Cycle. 123 3 7.1 The Development of Conceptions About Biological Oxidation 124 7.2 Modern Conceptions about Biological Oxidation 126 7.3 Oxidative Decarboxylation of Pyruvate 137 7.4 The Citric Acid Cycle 139 Chapter 8 Hormones 144 8.1 Features of Biological Action of Hormones 144 8.2 Classification of Hormones 146 8.3 Hypothalamic and Pituitary Hormones 155 8.4 Thymus Gland Hormones 161 8.5 Pineal Gland Hormones 161 8.6 Thyroid Hormones 162 8.7 Hormones That Regulate Calcium Metabolism 165 8.8 Hormones of Pancreas and Gastro-intestinal Tract 166 8.9 Hormones of Adrenal Medulla 169 8.10 Hormones of Adrenal Cortex 171 8.11 Sex Hormones 175 8.12 Eicosanoids 178 8.13 Cytokines 181 Chapter 9 Carbohydrate Metabolism 185 9.1 Carbohydrate Digestion 185 9.2 Absorption of Monosaccharides 186 9.3 Glycogen Metabolism and its Regulation 187 9.4 Aerobic and Anaerobic Oxidation of Glucose 191 9.5 Gluconeogenesis. Regulation of Gluconeogenesis and Glycolysis 199 9.6 Pentose Phosphate Pathway 204 9.7 Regulation of Blood Sugar 209 9.8 Disturbances of Carbohydrate Metabolism 211 Chapter 10 Lipid metabolism 221 10.1 Digestion and Absorption of Lipids 221 10.2 Synthesis of Fats 225 10.3 Transport Forms of Lipids 226 10.4 Lipid Metabolism in Adipose Tissue 230 10.5 Conversions of Glycerol 232 10.6 Oxidation of Fatty Acids 233 10.7 Ketogenesis and Ketolysis 237 10.8 Synthesis of Fatty Acids 240 10.9 Metabolism of Cholesterol 244 10.10 Disturbances of Lipid Metabolism 248 4 Chapter 11 Metabolism of Simple Proteins 255 11.1 Digestion and Absorption of Proteins 256 11.2 Tissue Proteolysis. Cathepsins 262 11.3 Common Pathways of Amino Acid Metabolism: Decarboxylation, Deamination, Transamination 263 11.4 Ammonia Detoxication. Urea Synthesis 273 11.5 Catabolism of Carbon Skeletons of Amino Acids. Gluco- and Ketogenic Amino Acids 281 11.6 Conversion of Amino Acids to Specialized Products 284 Chapter 12 Nucleic Acid Metabolism 299 12.1 Metabolism of Nucleotides 399 12.2 Synthesis of Nucleic Acids 310 12.2.1 DNA Replication 310 12.2.2 RNA Synthesis (Transcription) 314 12.3 Molecular Mechanisms of Mutations. DNA Repair 319 12.4 Recombinant DNA Technology. Application 322 Chapter 13 Synthesis of Protein ant Its Regulation 326 13.1 Biosynthesis of Proteins 326 13.2 Regulation of Protein Synthesis 332 13.3 Inhibitors of Transcription and Translation. Their Utilization in Medicine 335 Chapter 14 Blood 338 14.1 Physico-chemical Properties. Buffer Systems of Blood. Acid-base Balance. 338 14.2 Chemical Composition of Blood 345 14.3 Respiratory Function of Erythrocytes. Hemoglobin Metabolism 356 14.3.1 Respiratory Function of Erythrocytes 356 14.3.2 Hemoglobin Metabolism 363 14.4 System of Hemostasis 375 14.4.1 Blood Coagulation System 376 14.4.2 Anticoagulant System 386 14.4.3 Fibrinolytic System of Blood 387 Chapter 15 Immune Processes 393 15.1 Nonspecific Immune Defense Mechanism 395 15.1.1 Cellular Factors of Nonspecific Immunity 396 15.1.2 Humoral Factors of Nonspecific Immunity 399 15.2 Acquired (Adaptive) Immune System 406 5 15.2.1 Antibodies 407 15.2.2 Mechanisms Providing Variability of Antibodies 410 15.3 Simplified Diagram of Immune Response 411 15.4 Disturbances of Immune System 413 Chapter 16 Biochemistry of Kidney. Metabolism of Water and Minerals 419 16.1 Biochemistry of Kidney 419 16.1.1 Glomerular Filtration 420 16.1.2 Reabsorption and Secretion 421 16.1.3 Functions of Kidney 421 16.1.4 Physico-chemical Properties of Urine 424 16.1.5 Chemical Composition of Urine under Normal and Pathologic Conditions 426 16.2 Metabolism of Water and Minerals 428 16.2.1 Water Metabolism 429 16.2.2 Regulation of Water-salt metabolism 432 16.2.3 Role of Some Minerals 437 16.2.4 Water and Mineral Metabolism Disorders 441 Chapter 17 Biochemistry of Liver. Biotransformation of Xenobiotics 446 17.1 Functions of Liver 446 17.2 Role of the Liver in Carbohydrate Metabolism 446 17.3 Role of Liver in Lipid Metabolism 447 17.4 Role of Liver in Protein Metabolism 448 17.5 Bile Formation Function of Liver 448 17.6 Storage Function 449 17.7 Biotransformation Function 449 17.8 Ethanol Metabolism 452 Chapter 18 Nervous Tissue 456 18.1 Features of Biochemical Composition 456 18.2 Feature of Metabolism in Nervous Tissue 457 18.3 Synaptic Signal Transmission 460 18.4 Biochemistry of Neuromediators 464 Chapter 19 Muscle Tissue 476 19.1 Functions and Chemical Composition 476 19.2 Features of Metabolism 480 19.3 Energy Sources for Muscular Activity 482 19.4 Mechanism of Muscle Contraction 484 19.5 Features of Contraction of Smooth Muscle 488 6 19.6 Biochemical Changes in Muscle in Pathology 492 Chapter 20 Connective tissue 496 20.1 General Characteristic of Connective Tissue, Functions 496 20.2 Features of Chemical Composition of Connective Tissue 497 20.3 Features of Metabolism in Connective Tissue 504 20.4 Disorders of Connective Tissue 509 References 516 Index 517 7 LIST OF ABBREVIATIONSLIST OF ABBREVIATIONS A adenine CDP cytidine diphosphate ACAT acyl-CoA:cholesterol acyltransferase CSF Cys colony stimulating factor cysteine ACP acyl carrier protein ACTH adrenocorticotropic hormone DOPA dihydroxyphenylalanine ADH antidiuretic hormone DAG diacylglycerol ADP adenosine diphosphate DAP dihydroxyacetone phosphate AIDS aquired immunodeficiency syndrome DNA deoxyribonucleic acid Ala alanine Ea activation energy AMP adenosine monophosphate E0 redox potential value APC antigen presenting cell EF elongation factor apo apoprotein Arg arginine FAD flavin adenine dinucleotide Asn asparagine FADH2 reduced form of FAD Asp aspartic acid FFA free fatty acid ATP adenosine triphosphate FH2 dihydrofolate FH4 tetrahydrofolate C cytosine fMet formylmethionine cAMP cyclic adenosine monophosphate FMN FSH flavin mononucleotide follicle-stimulating hormone cGMP cyclic guanosine monophosphate G guanine CoA coenzyme A G0 standard free energy CoQ coenzyme Q, ubiquinone GABA γ-aminobutyric acid 8 GAG glycosaminoglycan G-1-P glucose-1-phosphate Gal-1-P galactose-1-phosphate G-6-P glucose-6-phosphate GAP glyceraldehyde-3-phosphate Gln glutamine Ka acid dissociation constant Glu Gly glutamic acid glycine Keq equilibrium constant of reaction Km Michaelis constant Hb hemoglobin α-KG α-ketoglutarate HbO2 oxyhemoglobin HDL high-density lipoprotein LCAT lecithin:cholesterol HGPRT Hypoxantine-guanine phosphoribosyl transferase LCK acylthransferase light chain kinase His histidine LDL low-density lipoprotein HIV human immunodeficiency virus Leu LH leucine luteinizing hormone HMG- CoA 3-hydroxy-3-methylglutaryl- CoA Lys lysine Hsp heat shock protein MHC major histocompatibility complex I inosine mRNA messenger RNA IDL intermediate-density lipoprotein N unspecific base of IF initiation factor nucleotide IFN interferon NAD nicotinamide adenine IG immunoglobulin dinucleotide IGF insulin-like growth factor NADH reduced form of NAD IL interleukine NADP nicotinamide adenine Ile isoleucine dinucleotide phosphate 9 NADPH reduced form of NADP S “synthesis” phase of cell cycle NCAM nerve cell adhesion molecule SAM S-adenosyl methionine Ser serine P high-energy phosphate STH somatotropic hormone P450 cytochrome P450 Pi inorganic phosphate T thymine PAF platelet activation factor T3 triiodothyronine PEP phosphoenolpyruvate T4 thyroxine PG prostaglandin TAG triacylglycerol Phe phenylalanine TCA tricarboxylic acid cycle PK proteinkinase TF transcription factor PKU phenylketonuria Thr threonine PLP pyridoxal phosphate TNF tumor necrosis factor PPi inorganic pyrophosphate TPP thiamine pyrophosphate Pro proline tRNA transfer RNA PRPP 5-phosphoribosyl-1- pyrophosphate Trp TSH tryptophan thyroid stimulating hormone Tyr tyrosine Q ubiquinone U uracil RNA ribonucleic acid UDP uridine diphosphate RNase ribonuclease rRNA ribosomal RNA Val valine VLDL very low-density lipoprotein S Svedberg unit 10 FOREWORDFOREWORD Biochemistry holds a key position in training medical students, and is one of the basic preclinical science subjects in medical universities. Through knowledge og biochemistry by medical students is very important for the understanding and maintenance of health and for the understanding and effective treatment of disease. Biochemistry is being transformed with astonishing rapidity. The material contained in the manual reviews the major advances in the static, dynamic and functional biochemistry. The material of manual is presented in 20 chapters. It includes structure and metabolism of basic classes of biomolecules (proteins, amino acids, nucleic acids, nucleotides, carbohydrates, lipids), regulation of metabolism and physiologic functions, biochemistry of enzymes, vitamins, blood, kidney, muscle, liver, immune, nervous, connective tissues. Modern conceptions of molecular biology and genetics, other complex topics are presented in brief and accessible form. Elucidation of molecular mechanisms of different classes of substances metabolism disorders, development of different diseases increases the clinical orientation of manual. It is imperative for understanding the causes and rational treatment of many diseases, two major areas of interest to physicians and other health care workers. 11 Chapter 1 STRUCTURE OF PROTEINS AND AMINO ACIDSChapter 1 STRUCTURE OF PROTEINS AND AMINO ACIDS Proteins are bioorganic high molecular nitrogen containing compounds, heteropolymers, which consist of amino acids residues combined by peptide bonds. Proteins are mostly prevalent from all the classes of biomolecules. They are involved to all cell components of microorganisms, plants, animals and inerstitial structures. 1.1 Functions of Proteins • Catalytic function. Biological catalysts are proteins. • Structural function. Proteins are included to biomembrane structure, form the base of cytoskeleton, intercellular matrix and certain specialized tissues. • Receptor function. Receptors are proteins. • Regulatory function. A lot of bioregulators (hormones, mediators, modulators) are proteins. • Transport function. Proteins provide intercellular and intracellular transfer of different substances. Also they carry chemical compounds in blood. • Contractive function. Proteins realize contraction of muscles, flagellums, cilia etc. • Protective function. Proteins provide the immune protection, prevent bleeding and intravessel thromb formation. 1.2 Proteinogenic Amino Acids. Proteins are high molecular substances, their molecular weight varies from some thousands till some millions atomic mass units or Daltons. A protein is the polymer in which the monomer units are amino acids. Amino acid is an organic compound that contains both an amino (-NH2) and a carboxyl (-COOH) groups. The amino acids found in proteins are always α-amino acids - that is, amino acids in which the amino group is attached to the α- carbon 12 atom of the carboxylic acid carbon chain. The general structural formula of an α- amino acid is: C CH2N H R O OH The R-group present in α-amino acid is called the amino acid side chain. The nature of these side chains distinguishes α-amino acids from each other. Side chain varies in size, shape, charge, acidity, functional group present, hydrogen-binding activity and chemical reactivity. Over 700 different naturally occurring amino acids are known, but only 20 of them, called standard amino acids, are normally present in proteins. Standard amino acid is one of the 20 α-amino acids normally found in proteins. These amino acids are called proteinogenic. Amino acids Acyclic: Cyclic - monoaminomonocarbonic - monoaminodicarbonic Carbocyclic Heterocyclic - diaminomonocarbonic - diaminodycarbonic With primary amino Imino acids group in the side chain Besides their classification by side-chain functional group amino acids are also classified by side-chain polarity. In this system there are four categories: (1) nonpolar amino acids, (2) polar neutral amino acids, (3) polar negatively charged amino acids, (4) polar positively charged amino acids (table 1.1). Table 1.1 Proteinogenic Amino Acids Name International symbol Structural formula pI Amino acids with non polar R Alanine Ala (A) H2N CH C CH3 OH O 6.02 13 Valine Val (V) H2N CH C CH OH O CH3 CH3 5.97 Leucine Leu (L) H2N CH C CH2 OH O CH CH3 CH3 5.98 Isoleucine Ile (I) H2N CH C CH OH O CH3 CH2 CH3 6.02 Methionine Met (M) H2N CH C CH2 OH O CH2 S CH3 5.75 Proline Pro (P) N H C HO O 6.10 Tryptophan Trp (W) H2N CH C CH2 OH O HN 5.88 Phenylalanine Phe (F) H2N CH C CH2 OH O 5.98 Amino acids with polar neutral R Glycine Gly (G) H2N CH C H OH O 5.97 Serine Ser (S) H2N CH C CH2 OH O OH 5.68 Threonine Thr (T) H2N CH C CH OH O OH CH3 6.53 Cysteine Cys (C) H2N CH C CH2 OH O SH 5.02 Tyrosine Tyr (Y) H2N CH C CH2 OH O OH 5.65 Asparagine Asn (N H2N CH C CH2 OH O C NH2 O 5.41 14 Glutamine Gln (Q) H2N CH C CH2 OH O CH2 C NH2 O 5.65 Amino acids with negatively charged R Aspartic acid Asp (D) H2N CH C CH2 OH O C OH O 2.97 Glutamic acid Glu (E) H2N CH C CH2 OH O CH2 C OH O 3.22 Amino acids with positively charged R Lysine Lys (K) H2N CH C CH2 OH O CH2 CH2 CH2 NH2 9.74 Arginine Arg (R) H2N CH C CH2 OH O CH2 CH2 NH C NH2 NH 10.76 Histidine His (H) H2N CH C CH2 OH O N NH 7.58 • Nonpolar amino acids contain one amino group, one carboxyl group and a nonpolar side chain. They are generally found inside of proteins, where there is limited contact with water. • Polar neutral amino acids contain one amino group, one carboxyl group, and a side chain that is polar but neutral. • Polar negatively charged amino acids contain one amino group and two carboxyl groups, the second carboxyl group being part of the side chain. In solution at physiological pH the side chain of a polar acidic amino acid has a negative charge; the side chain carboxyl group loses its acidic hydrogen atom. • Polar positively charged amino acids contain two amino groups and one carboxyl group, the second amino group being part of the side chain. In 15 solution at physiological pH the side chain of a polar basic amino acid has a positive charge; the side chain amino group accepts a proton. Properties of proteinogenic amino acids. • Chirality of amino acids. Four different groups are attached to one α-carbon atom in all of the standard amino acids except glycine, where the R group is a hydrogen atom. This means that the structure of 19 from 20 standard amino acids posess a chiral center. With few exceptions (in some bacteria), the amino acids found in nature and in proteins are L-isomers. Two of the 19 chiral standard amino acids, isoleucine and threonine, posess two chiral centers (table 1.1). With two chiral centers present, four stereoisomers are possible for these amino acids. However, only one of the L-isomers is found in proteins. • Acid-base properties. In neutral solution carboxyl groups have a tendency to lose protons (H+), producing a negatively charged species and amino groups have a tendency to accept protons (H+), producing a positively charged species. In neutral solution the –COOH group of an amino acid donates a proton to the –NH2 of the same amino acid and amino acid molecules have a structure: H2N H C COO- R -H+ H3 +N H C COO- R H3 +N H C COOH R +H+ Such a molecule is known as zwitterion, from the German term meaning “double ion”. A zwitterion is a molecule that has a positive charge on one atom and the negative charge on another atom. The net charge on a zwitterion is zero even though parts of the molecule carry charges. Zwitterion structure changes when the pH of a solution containing an amino acids is changed from neutral either to acidic (low pH) by adding an acid such as HCl or to basic (high pH) by adding a base such as NaOH. In an acidic solution, the zwitterion accepts a proton (H+) to form a positively charged ion. In basic solution, the –NH3 + of the zwiterion loses a proton and a negatively charged species is formed. 16 Thus, in solution three different amino acid forms can exist (zwitterions, negative ion, positive ion). The three species are actually in equilibrium with each other, and the equilibrium shifts with pH change. The overall equilibrium process can be represented as follows: H2N H C COO- R -H+ H3 +N H C COO- R H3 +N H C COOH R +H+ In acidic solution, the positively charged species on the right predominates; nearly neutral solutions have the middle species (the zwitterion) as the dominant species; in basic solution, the negatively species on the left predominates. Because of the extra site that can be protonated or deprotonated, acidic and basic amino acids have four charged forms in solution. COOH C HH3N CH2 COOH low-pH form (+1 charge) COO- C HH3N CH2 COOH COO- C HH3N CH2 COO- COO- C HH2N CH2 COO- moderately-low-pH form (zwitterion) neutral-pH form (-1 net charge) high-pH form (-2 net charge) OH- H3O+ OH- OH- H3O+ H3O+ The existence of two low-pH forms for aspartic acid results from the two carboxyl groups being deprotonated in different times. For basic amino acids, two high-pH forms exist because deprotonation of the amino groups does not occur simultaneously. The side-chain amino group deprotonates before the α-amino groups. Under the equilibrium of negative and positive charges amino acid molecule shows the isoelectric state. Individual for each amino acid pH level, under which amino acid has summary zero charge is called isoelectric point. • Ability to form amide bonds. Removal of the elements of water from the reacting carboxyl and amino groups leads to the formation of the amide bond. In amino acid chemistry, amide bonds that link amino acids together are given the specific name of peptide bond. A peptide bond is a bond between the carboxyl group of one amino acid and the amino group of another amino acid. 17 O C H C NH2R1 H C NH2 COOH + R2 HO CH C N CH O H R1 H2N COOH R2 + H2O peptide group Short to medium-sized chains of amino acids are known as peptides. A peptide is a sequence of amino acids up to 50 units in which the amino acids are joined together through amide (peptide) bonds. In all peptides, the amino acid at one end of the amino acid sequence has a free amino group, and the amino acid at the other end of the sequence has a free carboxyl group. The end with a free amino group is called the N-terminal end, and the end with a free carboxyl group is called the C-terminal end. By convention, the sequence of amino acids in a peptide is written with the N-terminal end amino acid at the left. The individual amino acids within a peptide chain are called amino acid residues. Peptide Bond Properties • Complanarity. Four atoms which form peptide bond (-CO-NH-) are situated in the same geometric flatness. • Oxygen of carbonyl group and hydrogen of NH- group are situated in trans- position. • Length of bond between carbon from CO group and nitrogen from NH is equal 1,032 nm. Peptide bond is intermediate between single and double bond (that is this bond has the partial double bond character, therefore rotation around this bond is impossible). These restriction facilitate the specific configuration of chains. • Keto-enol thautomery. There are two conformations of peptide bond – ketone and enol. The resonance structure formation is provided by coupling free p- electrone pair of double bond C=O. C N O H C N OH • Peptide bond is able to form hydrogen bonds. 18 N H CO 1.3 Levels of Protein Structure Proteins are polypeptides that contain more than 50 amino acid residues. Primary structure is the unique linear sequence of amino acid residues in a polypeptide chain. The name of a peptide is made up of the name of the first N-terminal amino acid bearing a free NH2-group (with the ending -yl) and of the names of amino acids added in succession (likewise each with ending -yl); the formulation ends the full name of the C-terminal amino acid with a free COOH group. For example: glycyl-phenylalanyl-serine or Gly-Phe-Ser: H2N CH C H O HN CH C CH2 O HN CH C CH2 OH O OH Primary structure is very stable. It provides the stability of protein molecules. It predetermines all the other levels of protein organization. • Secondary structure is the configuration of a polypeptide chain due to formation of hydrogen bonds between components of peptide bonds. Polypeptide chain strives to configuration with maximum possible number of hydrogen bonds. But possibilities of spatial packing of polypeptide chain are limited by partial double bond character of peptide bond and impossibility of rotation around of this bond. Therefore polypeptide chain has specific conformation. The following types of secondary structure are known: - α-coil or α-helix; - β-pleated sheet; - helix of collagen (triple helix); - irregular conformations (disordered regions). 19 • α-Helix is the conformation which is generated at the space rotation of polypeptide chain by the hydrogen bonds, which are formed between C=O and NH groups distant one to another at four amino acid residues. Hydrogen bonds of α-helix are directed parallely to the molecular axis. The direction of α-helix rotation in natural proteins is right. Geometric parameters of α-helix: radius – 0,25 nm, step – 0,54 nm, length of displacement to one amino acid residue – 0,15 nm, one revolution of α-helix is equal to 3,6 amino acid residues. Several amino acids such as Pro, Gly, Glu, Asp, Arg counteract of α-helix formation or destabilize it. Figure 1.1 The α-helix • β-Pleated sheet is the structure similar to the folded lay. It is formed from zigzag-liked unwrapped nearly situated polypeptide chains. β-Pleated sheet structure is also formed by the hydrogen bonds, which are formed between C=O and NH groups of neighbouring chains. Figure 1.2. β-pleated sheet • The triple helix is another secondary structure for proteins. This structure involves three coiled polypeptide chains wound around each other about a common axis to give a rope-like arragement. The interwining of the three polypeptide chains and some cross-linking between chains involving covalent bonds hold the triple helix together. 20 Collagen, the structural protein of connective tissue, which contains 33% of glycine and 21 % of hydroxyproline, has a triple helix structure. Super-secondary structure. The α-helix and β-pleated sheets are connected together by unstructured polypeptides. The existence for some proteins of super- secondary structure has been found by means of X-ray crystallography methods. Figure 1.3. Supersecondary structure. These proteins are called domain proteins. These proteins contain to a conciderable extent isolated globules-domains. These globules in domain proteins are formed by the same single polypeptide chain. Tertiary structure of domain proteins is the packing different domains each to another. Domains usually perform different functions. Functional properties of domain proteins are like to those of oligomeric ones. • Tertiary structure is the overall three-dimentional shape that results from the attractive forces between amino acid side chains (R groups) that are widely separated from each other within the chain. Four types of attractions contribute to the tertiary structure of a protein: covalent disulfide bonds, ionic bonds (salt bridges), hydrogen bonds and hydrophobic interactions. - Disulfide bonds (-S-S-), the strongest of the tertiary structure interactions, result from –SH groups two cysteine molecule residues, which are included to the same or different polypeptide chains. This type of interaction is the only one of the four tertiary-structure interactions that involves a covalent bond. - Ionic bonds, also called salt bridges, always involve amino acids with charged side chains. These amino acids are the acidic and basic amino acids. The two R-groups, one acidic and one basic, interact through ion-ion interactions. 21 - Hydrogen bonds can occur between amino acids with polar R groups. A variety of polar side chains may be involved especially those that possess the following functional groups: -OH, -NH2, -C-OH, -C-NH2. O O Hydrogen bonds are relatively weak and are easily disrupted by changes of pH and temperature. - Hydrophobic interactions result when two nonpolar side chains are close to each other. Anthough hydrophobic interactions are weaker than hydrogen and ionic bonds, they are a significant force in some proteins because there are so many of them. Depending on the shape and features of three-dimensional shape there are globular and fibrillar proteins. Globular proteins have roughly spherical shape. The ratio between long and short axes varies from 1:1 till 50:1. Globular proteins are built from one or some polypeptide chains, which are tightly packed. Figure 1.4 Tertiary structure. In aqueous solution, globular proteins usually have their polar R groups outward toward the aqueous solvent (which is also polar), and their non polar R groups inward (away from the polar water molecules). The non polar R groups then interact with each other. - Fibrillar proteins show the linear shape. They usually form multimolecular complexes – fibrils, which consist of some parallel polypeptide chains. Fibrillar proteins are structural components of connective and other tissues. A lot of fibrillar proteins are formed by superspiralization. • Quaternary structure is the highest level of protein organization. It is found in proteins that have two or more polypeptide chains. These multichain 22 proteins are often called oligomeric proteins. Quaternary structure is formed by aggregation of some polypeptide chains or protomers, each of them shows characteristic ordered conformation. Subunits are combined by non covalent bonds. This provides easy dissociation under change of physico-chemical properties of medium. Physico-chemical Properties of Proteins The most characteristic physico-chemical properties inherent in proteins are: high viscosity in solution; low diffusion; pronounced swelling ability; optical activity; mobility in electric field; low osmotic and high oncotic pressures; ability ro absorb UV light at 280 nm wave-length (this property which is attributable to the occurence of aromatic amino acids in proteins, is used of for protein quantitative determination). The most of physico-chemical properties of proteins are provided by their acid-base properties and high molecular mass. • Acid-base properties. Due to the presence of high amount of ionogenious groups proteins are amphoteric electrolytes and form amphions in the water solutions, which charge depends on the amino acid composition and pH of medium. Presence of charge provides the protein mobility in the electric field. Electrophoretic activity depends on the charge and molecular mass and allows the using electrophoresis to separate protein mixture. pH changing can cause the state, when summary charge of protein molecule is zero (isoelectric state). The level of pH, when summary charge of protein molecule is zero is called isoelectric point (pI). At the isoelectric point, the proteins are the least stable in solution and are prone to an easy precipitation. • Solubility of proteins in different physico-chemical mediums depends on the presence of polar or non polar amino acid residues. The increase of metal cations or ammonium concentrations in the solution leads to dehydration and sedimentation of protein. 23 • Denaturation is the loss of spatial conformation, characteristic to the native protein molecule with the resultant loss of their native properties. Denaturation occurs under action of hard physical and chemical factors. The mechanism of such factors influence is based on destruction of weak bonds, which stabilize the spatial conformation. Figure 1.5. Protein denaturation. • Interaction with different chemical ligands. The presence on molecular surface of different active functional groups provides the protein ability to combine with various chemical ligands (low and high-molecular compounds). The binding with ligands can be the step of transport, regulatory or catalytic functions realizing. 1.4 Protein Classification Proteins Simple Conjugated - albumins - globulins Protein part Non-protein part - histones (apoprotein) (prosthetic group) - prolamines - protamines - glutelins Table 1.1 Simple Proteins Group Localization Biological role Features of structure and composition Histones. Molecular mass is 11 – 22 kDa Cell nuclei (deoxyribo- nucleo- proteins) They provide the formation and function of nuclear chromatin, regulation of genetic information transduction Bacis amino acids are prevalent: Lys, Arg, His Albumins. Molecular mass ~ 70 kDa. In organs and tissues: blood, muscles. They keep oncotic pressure, transport hormones, fatty acids, bilirubine, biogenic elements, drugs, form protein reserve, play plastic, detoxification roles. Acidic amino acids are prevalent: Glu, Asp. They contain 15% of Leu, 1% of Gly. Globulins. Blood, They perforn transport, Contain 3% of Gly. 24 Molecular mass is 150 kDa. muscles, lymph protective functions, take part in blood clotting Similar to albumin, but contain low amount of Asp, Glu. The distinctive definitions “albumins and globulins” have been suggested in reference to the respective solubility or insolubility of these proteins in distilled water and half-saturated aqueous (NH4)2SO4 solution. Globulins are soluble only in dilute saline solutions and insoluble in other media. It should be noted that some classical globulins of blood serum (for example, β-lactoglobulin) are soluble in 50% (NH4)2SO4 solution. The different solubility of blood serum albumins and globulins is widely used in clinical practice for their fractionation and quantitive determination. Conjugated Proteins Conjugated proteins consist of protein part, which is called apoprotein, and non-protein part or prosthetic group. Apoprotein and prosthetic group can be combined together by covalent or noncovalent bonds. According to the chemical nature of proshtetic group conjugated proteins are divided to: • Glycoproteins. The prosthetic group in glycoproteins is represented by carbohydrates and their derivatives bound quite tightly to the protein moiety of glycoprotein molecules. Protein complexes with high molecular heteropolysaccharides are called proteoglycans. • Lipoproteins are complex proteins, which protein part is combined with lipids. • Nucleoproteins are proteins combined with nucleic acids (DNA or RNA). Nucleoproteins are supramolecular complexes, which form cell organells (ribosomes), chromatin. • Chromoproteins are proteins with colored prosthetic group. Table 1.2 Chromoproteins Groups Biological role Prosthetic group Flavoproteins (FAD, FMN) Participation in the biological oxidation, oxido-reductive processes Isoalloxazine derivatives 25 Rhodopsin Protein of visual purple, participation in visual act Retinal Hemoproteins: hemoglobin, cytochromes, catalase, peroxidases, myoglobin Respiratory, transport, electron transfer, catalytic functions Iron-containing protoporphyrines (heme) • Metaloproteins contain metal, which is not involved to the metaloporphyrine complex. • Phosphoproteins are proteins, which contain phosphate residue combined by phosphodiesteric bond with hydroxyl group of serine, threonine or tyrosine of polypeptide chain. 26 Tests for Self-control 1. In proteins the α-helix and β-pleated sheet are examples of: A. Primary structure B. Secondary structure C. Tertiary structure D. Quaternary structure E. None of the above mentioned 2. Which amino acids contain negatively charged R-group? A. Leucine and asparagine B. Glutamine and glycine C. Glutamic and aspartic D. Arginine and valine E. Glycine and serine 3. Which amino acids contain positively charged R-group? A. Leucine and asparagine B. Glutamine and glycine C. Glutamic and aspartic D. Arginine and valine E. Arginine and histidine 4. Which chemical feature is not characteristic for peptide bond? A. Complanarity B. Keto-enol thautomery C. Ability to form hydrogen bonds D. Mutarotation E. Intermediate length between single and double bonds 5. Denaturation is: A. The loss of spatial conformation, characteristic to the native protein molecule B. Destruction of peptide bonds C. Replace of C-end D. Replace of N-end E. Deamination of amino acids 6. Protein part of the complex protein is called: A. Lipoprotein B. Apoprotein C. Prosthetic group D. Phosphoprotein E. Peptide 27 Chapter 2 CARBOHYDRATES AND THEIR DERIVATIVESChapter 2 CARBOHYDRATES AND THEIR DERIVATIVES Carbohydrates are bioorganic substances, which by their chemical structures are aldehydo- or ketoderivatives of polyatomic alcohols or polyhydroxyaldehydes and polyhydroxyketones. Carbohydrates, which are cannot be hydrolyzed are simple carbohydrates or monosaccharides. Carbohydrates, which are hydrolyzed with monosaccharides formation, are called polysaccharides or oligosaccharides. Carbohydrates have an empirical formula (CH2O)n. But many polysaccharides, however, especially those which have a structural role, do not satisfy this formula in all respects because their monomer components are modified and include amino sugars, deoxy sugars and sugar acids. Nonetheless, they are called carbohydrates. In plants, glucose is synthesized from carbon dioxide and water by photosynthesis and stored as starch or is converted to the cellulose of the plant framework. Animals can synthesize some carbohydrates from non carbohydrates compounds, but the bulk of animal carbohydrate is derived ultimately from plants. 2.1 Monosaccharides and Their Derivatives Monosaccharides, the simplest carbohydrates, are aldehydes or ketones that have two or more hydroxyl groups. According to the carbon atoms amount monosaccarides are divided to trioses, tetroses, pentoses, hexoses, heptoses etc. Hexoses and pentoses are most prevalent monosaccarides as participants of metabolism or components of other biomolecules. The smallest ones, for which n=3, are glyceraldehyde and dihydroxyacetone. They are trioses. Glyceraldehyde is an aldose because it contains an aldehyde group, whereas dihydroxyacetone is a ketose because it contains a keto group. 28 Figure 2.1 D-glyceralsehyde (left), L-glyceralsehyde (middle), dihydroxyacetone (right) Glyceraldehyde has a single asymmetric carbon. Thus, there are two stereoisomers: D-glyceraldehyde and L-glyceraldehyde. The prefixes D and L designate the absolute configuration. They do not differ in physical properties, but each rotates the plane of polarized light in the opposite direction of each other. The direction of rotation cannot be inferred from the absolute configuration (symbols D and L), and is recorded by the symbols “plus” and “minus”, or some times by symbols d and l, which are confused since they have no relation to the D and L forms of the molecule. Sugars with 4, 5, 6 and 7 carbon atoms are called tetroses, pentoses, hexoses and heptoses. Two common hexoses are D-glucose (an aldose) and D-fructose (a ketose). For sugars with more than one asymmetric carbon atom, the symbols D and L refer to the absolute configuration of the asymmetric carbon farthest from the aldehyde or keto group. In general, a molecule with n asymmetric centers has 2n stereoisomeric forms. For aldotrioses n is equal to 1, and so there are 2 stereoisomers, D- and L- glyceraldehyde. They are enantiomers of each other, that is, so called “mirror images”. Figure 2.2 D- and L-glucose. The six-carbon aldoses have four asymmetric centers, and so there are 16 stereoisomers and 8 of them belong to the D-series. D-Glucose, D-mannose and D- 29 H C C CH2OH H OH O H C C CH2OH H OH O CH2OH C CH2OH O 1CHO 2C HHO 3C OHH 4C HHO 5C HHO 6CH2OH CHO C OHH C HHO C OHH C OHH CH2OH L-glucose D-glucose galactose are abundant six-carbon aldoses. D-glucose and D-mannose differ only in configuration at C-2. D-Sugars differing in the configuration a single asymmetric center are epimers. Thus, D-glucose and D-mannose are epimers at C- 2; D-glucose and D-galactose are epimers at C-4. The predominant forms of glucose and fructose in solution are not open- chains. Rather, the open-chain forms of these sugars form rings. The C-1 aldehyde in the open-chain form of glucose reacts with the C-5 hydroxyl group to form an intramolecular hemiacetal. The resulting sixmembered sugar ring is called pyranose. The C-2 keto group in the open-chain form of fructose can react with the C-5 hydroxyl group to form an intramolecular hemiketal. This fivemembered sugar ring is called furanose. An aditional asymmetric center is created when glucose cyclizes. Carbon-1, the carbonyl carbon atom in the open-chain form, becomes an asymmetric center in the ring form. Two ring structures can be formed, namely: α-D-glucopyranose and β-D-glucopyranose. The designation α means that the hydroxyl group attached to C-1 is below the plane of the ring; α means that it is above the plane of the ring. The C-1 carbon is called the anomeric carbon atom, and so the α and β forms are anomers. Sugars containing free hydroxyl group of anomeric carbon atom are the reducing sugars. They reduce indicators such as cupric ion (Cu2+) complexes to the cuprous form (Cu+). • Pentoses. Biologically important are: - aldopentoses: ribose, 2-deoxyribose, arabinose, xylose; - ketopentoses: ribulose, xylulose. Figure 2.3 β-D- ribofuranose Ribose is aldopentose, which in β-furanose form is included to nucleotide structure, coenzymes (NAD, NADP, FAD, FMN), glycosides, antibiotics, RNA. 30 2-deoxy-D-ribose differs from ribose by the absence of oxygen atom in the second position. It is involved to the deoxiribinucleotides structure. Figure 2.4. 2-Deoxy D-β-ribofuranose. Xylose is the source for synthesis of alcohol xylit, which is used in medicine. • Hexoses. They are divided to: - aldohexoses, for example glucose, galactose, mannose, fucose etc.; - ketohexoses, for example fructose. Glucose (grapes sugar, dextrose) is very frequent monosaccharide in the nature. It is the structural component of disaccharides and homopolysaccharides. Figure 2.5 α- (left ) and β-( right)-D-glucose. OH OH H OH H OHH OH CH2OH H CHO C OHH C HHO C OHH C OHH CH2OH O H HO H HO H HO OHH H OH Figure 2.6 α-D-glucose. It can exist in cyclic and linear forms. Position of H and OH groups around the fifth carbon atom causes D- or L- isoforms generation. Namely D-glucose is situated in the living been. Cyclic form can exist in α- or β- isoforms due to the position of H and OH groups around the first carbon. Galactose (milk sugar) is involved to the disaccharide lactose structure, which can be taken from the milk. Galactose is part of hetepolysaccarides (glycosaminoglycans or mucopolycaccharides) of animal tissues. 31 H OH H CH2OH OH H H O 1 23 4 5 H OOH H H OH H OHH OH CH2OH H OH OH H OH H OHH OH CH2OH H OH OH H OH OH HH OH CH2OH H a-D-galactose a-D-glucose a-D-mannose Figure 2.7 Epimerisation of glucose. Mannose is the structural component of polysaccharide mannan, which is the component of plant glycoproteins. In the animal organism mannose is involved to oligosaccharide part of glycolipids and glycoproteins of membrane and biological liquids. Product of mannose reduction – six-atomic alcohol mannitol (mannit) is used in medicine as osmotic diuretic and for replace of sucrose in the diet of patients with diabetes mellitus. Fructose is involved to disaccharide sucrose structure. It is monomer of polysaccharide inulin. OH OH CH2OH OH OH HOH H H H a-D-fructopyranose OH CH2OH H CH2OH OH H H OH O a-D-fructofuranose Figure 2.8 Fructose. Sugars are joined to alcohols and amines by glycosidic bonds. The crucial biological importance of N-glucosidic linkage is evident in such central biomolecules as nucleotides, RNA and DNA. N-glycosidic linkages in virtually all naturally-occuring biomolecules have the β -configuration. • Phosphorylated sugars are another important class of derivatives. They are key intermediates in energy generation and biosyntheses. In fact, one of the strategies of glycolysis is to form three-carbon intermediates that can transfer their phosphate groups to ADP to achieve an ATP synthesis. Phosphorylation also serves to make sugars anionic. They can have strong electrostatic interactions with the active site of an enzyme. The negative charge 32 contributed by phosphorylation also prevents these sugars from spontaneously crossing lipid bilayer membranes. Phosphorylation helps to retain biomolecules inside cells. Many times phosphorylated derivative of ribose plays a key role in the biosyntheses of purine and pyrimidine nucleotides as well. • Amino derivatives of monosaccharides. The mostly prevalent are two amino derivatives of hexoses – D-glucose and D-galactose – hexosamines. OH OH H OH H +NH3H OH CH2OH H Figure 2.9. Glucosamine. N-acetyled derivatives of hexosamines are frequently observed into heteropolysaccharides composition. Figure 2.10. N-acetyl-D-glucosamine (left) and N-acetyl-D-galactosamine (right). • Neuraminic and sialic acids. Neuraminc acid is biological important substance. It is derivative of the monosaccharide ketononose (nonulose). In organism neuraminic acid is present as N- and O-acyl-derivatives known as sialic acids. Neuraminic and sialic acids are structural components of membrane glycolipids, glycoproteins, and proteoglycans of biological fluids, mucus, connective tissue. Figure 2.11. N-acetyl-neuraminic acid. 33 • Aldonic acids are the products of aldehyde carbon oxidation of aldose. For example, gluconic acid, which as phosphate ester is formed in pentose phosphate pathway. • Uronic acids are the products of C-6 hydroxyl group oxidation of aldose. Acids, which are formed under glucose and galactose oxidation (glucuronic and galacturonic acids), are structural components of heteropolysaccharides. L- Iduronic acid is the structural component of heparin, dermatansulfates of connevtive tissue. Free glucuronic acid plays very important role in detoxification in animal organism. OH OH H OH H OHH OH COO- H OH OH H OH H OHH OH H COO- Figure 2.12. α-D-glucuronate (left) and β-L-iduronate (left). • Glycosides are the product of monosaccharide condensation with alcohols or phenols. They are formed by interaction of hydroxyl group of hemiacetal carbon atom of monosaccharide with OH group of alcohol (phenol). Plant steroid-containing glycosides show cardiotonic action and are used in medical practice as heart glycosides. 2.2 Oligosaccharides Oligosaccharides are carbohydrates that contain from two to ten monosaccharide units. Disaccharides are the most common type of oligosaccharides. Disaccharides consist of two monosaccharides joined by an O-glycosidic bond. Three highly abundant disaccharides are sucrose, lactose and maltose. Lactose (β-D-galactosyl-1.4-α-D-glucose) is milk sugar, consists of galactose and glucose residues, combined by 1.4 glycosidic bond. Lactose is important nutrient for human. 34 Sucrose (α-D-glucosyl-1.2-β-D-fructose) is one from the most spread in the nature and practically important disaccharide, which is situated in the plant stalks, roots, tubers. Maltose (α-D-glucosyl-1.4-α-D-glucose), malt sugar is disaccharide, which consists of two glucose molecules residues. Maltose is formed in the digestive channel from starch. OH OH H H OHH OH CH2OH H OH H OH H OHH OH CH2OH H O 1 23 4 5 6 1 23 4 5 6Maltose O-a-D-glucopyranosyl-(1--4)-a-D-glucopyranose OH OH H H OHH OH CH2OH H O 1 23 4 5 6 Sucrose O-a-D-glucopyranosyl-(1--2)-B-D-fructofuranose OH OH H H OHH OH CH2OH H OH H OH H OHH OH CH2OH H1 23 4 5 6 1 23 4 5 6Lactose O-B-D-galactopyranosyl-(1--4)-B-D-glucopyranose CH2OH HCH2OH OH H H OH O 2 1 3 4 5 6 O Figure 2.13. Most important disaccharides. Plant products contain trisaccharide raffinose, tetrasaccharide stachyose. 2.3 Polysaccharides Polysaccharides are carbohydrates made up of many monosaccharide units. Polysaccharides Homopolysaccharides Heteropolysaccharides consist of one type of consist of two and more monosaccharides types of monosaccharides 35 Homopolysaccharides - Starch is the plant polysaccharide, which consists of two fractions – amylose and amylopectin (15 – 25 and 80 – 85 % of starch mass respectively). Amylose is linear polysaccharide, with 200 – 1000 monomers (glucose residues), which are combined by α-1,4 glycosidic bonds (molecular mass ~ 40 – 160 kDa). Amylose homopolymers form spiral structures, each revolution involves six glucose molecules. Figure 2.14. Starch structure Amylopectin is the branched polysaccharide with molecular weight ~ 1 – 6 millions Da. The main chain of amylopectin is formed by α-1.4 glycosidic bonds; branching is generated by α-1.6 glycosidic bonds. Between branching points 20 – 30 glucose residues are situated. The sources of starch are bread, potato, beans. - Glycogen is animal homopolysaccharide with molecular weight ~ 100 millions Da. The chemical structure of glycogen is the similar to starch amylopectin, but has more branched molecules. The linear parts of main chain contain 6 – 12 glucose residues, combined by α-1.4 glycosidic bonds, branching is formed by α-1.6 glycosidic bonds. Glycogen forms intracellular granules. - Cellulose is homopolysaccharide, which is the main structural component of plant cell wall. Cellulose molecules are unbranched chains, which consist of glucose residues combined by β-1.4 glycosidic bonds. 36 - Dextran is branched polysaccharide of yeasts and bacterias. The main type of bond is α-1.6 glycosidic one. Branching is formed by α-1.2, α-1.3 or α-1.4 bonds. Dextrans are used in medicine as plasma and blood changers (Polyglucin, Reopolyglucin). Figure 2.15. Glycogen structure. - Inuline is plant polysaccharide, which consists of β-D-fructose residues, combined by β-1.2 glycosidic bonds, molecular mass is not more than 6 kDa. - Pectin is polygalacturonic acid derivative, which consists of α-D- galacturonic acid residues, combined by α-1.4 glycosidic bonds. Heteropolysaccharides Heteropolysaccharides consist of two and more types of the different monomers. Glycosaminoglycans are the most important heteropolysaccharides in the physiology. Glycosaminoglycans are polymers, which form interstial matrix of connective tissue. They are polyanion molecules. Some monosaccharide components contain acidic group – carboxyl or sulfate group, which provides high hydrophility. All glycosaminoglycans make their functions in the complex with proteins. Covalent complexes are called proteoglycans. Table 2.1 Structural Components of Glycosaminoglycans Glycosaminoglycans Composition of disaccharide unit Hyaluronic acid D-glucuronate + N-acetylglucosamine Chondroitin sulfate D-glucuronate + N-acetylgalactosamine sulfate 37 Dermatan sulfates D-iduronate (or D-glucuronate )+ N- acetylgalactosamine sulfate Keratan sufate D-galactose + N-acetylglucosamine sulfate Heparin and heparan sulfate D-glucuronate (or D-iduronate) + N-acetylglucosamine sulfate OH H H H OH COO- H OOSO3 - O H H H O CH2OH H O NH CO CH3 O OH Chondroitin-4-sulfate nB-glucuronic acid N-acetylgalactosamine sulfate OH H H H OH COSO3 - H OH H H H OH H COO- O OSO3 - O O NH SO3 - Heparin nsulfated glucosamine sulfated iduronic acid Figure 2.16. The main heteropolysaccharides. - Hyaluronic acid is linear polysaccharide, where D-glucuronic acid and N- acetylglucosamine are combined by β-1.3 glycosidic bonds; some single fragments are recombined by β-1.4 glycosidic bonds. Hyaluronic acid has the highest molecular weight in comparison with other glycosaminoglycans (105 – 107 Da). High amount of COO- forms the negative charge of molecule, provides water and Na+ ions keeping. Hyaluronic acid is present in bacteria and is widely distributed among various animal's tissues, including synovial fluid, the vitreous body of eye, cartilage, and loose connective tissue. The molecules coil and entwine to make a very firm gel at a very low concentration (0,1 %). The gell excludes other large molecules and also microorganisms, so that the rate of spread of bacterial infection is hindered. Many microorganisms secrete a hyaluronidase, which by shortening the average chain length of the polymer, greatly reduces the viscosity of the gel, and the secretion of hyaluronidase by certain tumour cells may correlate with the ability of these cells to metastasize. 38 OH H H H OH CH2OH H OH H H H OH CH2OH H O NH CO CH3 O O NH CO CH3 Chitin nN-acetylglucosamine N-acetylglucosamine OH H H H OH COO- H OH OH H H H O CH2OH H O NH CO CH3 O OH Hyaluronic acid nB-glucuronic acid N-acetylglucosamine - Chondroitin sulfates are important structural components of cartilages. Molecular mass is about 10 – 60 kDa. Disaccharide fragments consist of D- glucuronate and N-acetylgalactosamine sulfate combined by β-1.3 glycosidic bonds. - Keratan sulfates I and II consist of repeating galactose-N-acetylglucosamine or occasionasly of galactose. Type I is abundant in cornie and type II is found along with chondroitin sulfate attached to hyaluronic acid in loose connective tissue. - Dermatan sulfate is widely distributed in animal tissues. It consists of disaccharide units which consist of D-iduronic or D-glucuronic acids (iduronic acid is prevalent, combined with N-acetyl galactosamine). - Heparan sulfates are glycosaminoglycans, which are present on the surface of animal cells. They consist of D-glucuronic or D-iduronic acids combined with N-acetylglucosamine sulfate by β-1.4 glycosidic bonds. - Heparin is glycosaminoglycan, which is synthesized by connective tissue cells, and counteracts blood clotting (anticoagulant). Similarly to the heparan sulfates heparin chains contain disaccharide units, which consist of D-glucuronic or D-iduronic acids (iduronic acid is prevalent) combined with N- or O-glucosamine sulfate or N-acetylglucosamin by β-1.4 glycosidic bonds. Heparin and heparan sulfates have the common precursor as not sulfatized polysaccharide chain of heparin proteoglycan. Functions of Proteoglycans • Structural function. They are found in every tissue of the body, mainly in the extracellular matrix of “ground substance”. They are associated with each other and also with other major structural components of the matrix collagen and elastin. Some of them interact with certain adhesive proteins, such as fibronectin and laminin, which play the important role in the adhesion of cells to the extracellular matrix, fixation of basal membrane etc. 39 • Regulation of glomerular filtration. GAG heparin and heparan sulfate are components of basal membrane of renal glomerulars. Basal membrane regulates the passage of large molecules across the glomerulus into the renal tubule. The pores in the glomerular membrane are large enough to allow molecules up to about 8 nm to pass through. Albumin is smaller than this pore size, but it is prevent from passing through easily by the negative charges of heparan sulfate. These negative charges repel albumin which is negatively charged at the pH of blood. • Regulation of water-salt metabolism. The GAGs present in the proteoglycans are polyanions and hence bind polycations and cations such as Na+. This latter ability attracts water by osmotic pressure into the extracellular matrix. • Supporting the turgor of extracellular matrix. • Protective function. GAGs form gel at relatively low concentration, therefore they restrict the passage of large macromolecules into the extracellular matrix. • Regulation of permeability (hyaluronic acid). • They provide the cell migration during morphogenesis and wound repair (hyaluronic acid). • They provide compressibility. • They participates in osteogenesis (chondroitin sulfate). • They play a critical role in corneal transparency (heparan sulfate and dermatan sulfate). • Heparin is important anticoagulant. • Heparin activates lipoprotein lipase. • They are components of synaptic vesicles. 2.4 Functions of Carbohydrates • Energy function. Carbohydrates provide 60% of energy which is needed to organism. Oxidation of 1 gram of carbohydrates leads to liberation of 17,2 kJ (4,1 kcal). • Reserve function. Glycogen is a major storage form of carbohydrates in animals. Liver contains ~ 100g of glycogen, muscles – 200 – 250 g. 40 • Structural function. They are included into lipids, DNA, RNA, cell membrane, receptors. • Protective function. Participation of carbohydrate components of immunoglobulins in supporting of immunity. • They participate in performing of nervous impulse (glycolipids gangliosides). • They provide cell recognition, adhesion. • Some of them are anticlotting factors. 41 Tests for Self-control 1. Examples of hexoses are: A. Glucose, fructose, galactose B. Glucose, galactose, arabinose C. Ribose, erythrose, fructose D. Mannose, fructose, xylose E. Glucose, allose, ribose 2. Milk sugar consists of: A. Two glucose residues B. Glucose and fructose C. Galactose and fructose D. Galactose and glucose E. Two galactose residues 3. Which monosaccharide is included in the structure of DNA?: A. Glucose B. Xylulose C. Ribose D. Deoxyribose E. Ribulose 4. Which chemical bond presence provides the branching of starch and glycogen molecules? A. α-1.4 glycosidic B. α-1.3 glycosidic C. α-1.6 glycosidic D. β-1.4 glycosidic E. β-1.6 glycosidic 5. Which of the below mentioned carbohydrates is heteropolysaccharide? A. Starch B. Glycogen C. Maltose D. Heparin E. Cellulose 42 Chapter 3. STRUCTUREChapter 3. STRUCTURE AND FUNCTIONS OF LIPIDS AND FUNCTIONS OF LIPIDS Lipids are the chemical substances, which are insoluble in water and other polar solvents and soluble in non polar (hydrophobic) liquids. 3.1 Functions of Lipids • Energy function (1g – 9,3 kcal, 38,9 kJ). • Reserve function. Lipids form the energy reserve (10 kg – 30 – 40 days). • Fats serve as thermal insulators in subcutaneous tissues and around certain organs. • Nonpolar lipids act as electrical insulators allowing rapid propagation of depolarization waves along myelinated nerves. • They are included in the membrane structure therefore they influence on the membrane permeability and transmission of nervous impulse. • They are necessary for absorption of fat-soluble vitamins. • They perform the protective function. • They are precursors of important biological active substances: a) polyunsaturated fatty acids are precursors of Eicosanoids Prostanoids Leukotrienes - prostaglandins - prostacyclins - tromboxanes D3 b) Cholesterol steroid hormones bile acids c) phospatidyl- inositol 4,5-bisphosphate diacylglycerol inositol triphosphate Second merssengers 43 • They are important source of endogenious water (100 g lipids → 107 ml of water). 3.2 Common Characteristic of Lipids. Fatty Acids. By their chemical structure the main part of lipids is the complex esters of highest carbonic acids and alcohols. Some classes of lipids also involve phosphates, nitrogen compounds, carbohydrates etc. • Fatty acids. Fatty acid composition of lipids is the main sign, which provides their physico-chemical and biological properties. The carbon atom amount and the length of chain, range of saturation of fatty acids determine the consistency (liquid, solid) and surface activity, namely, the ability to form complexes with proteins and finally micelle, bilay, matrix of biological membranes. Usually lipids of human organism involve to their structure fatty acids with even number of carbon atoms, which consist of from 12 till 24 carbon atoms, prevalently from 16 C till 20 C, most commonly, an unbranched chain. Palmitic acid is prevalent saturated acid and oleic acid is prevalent unsaturated fatty acid. The high amount of oleic acid in lipids determines the liquid state of human body fats, melting temperature of which is about 10 -15oC. Role of Polyunsaturated Fatty Acids: • they are precursors of important biological active substances; • they support the liquid state of cell membranes; • they normalize the cholesterol metabolism; • they prevent the fatty liver; • antiradiation effect. 44 Table 3.1 The Main Fatty Acids Code index Structure Systematic name Trivial name Saturated fatty acids C12:0 CH3(CH2)10COOH n-Dodecanic Lauric C14:0 CH3(CH2)12COOH n-Tetradecanic Myristic C16:0 CH3(CH2)14COOH n-Hexadecanic Palmitic C18:0 CH3(CH2)16COOH n-Octadecanic Stearic C20:0 CH3(CH2)18COOH n-Eicosanic Arachidic C22:0 CH3(CH2)20COOH n-Docosanic Behenic C24:0 CH3(CH2)22COOH n-Tetracosanic Lignoceric Monounsaturated fatty acids C16:1 CH3(CH2)5CH=CH(CH2)7COOH cis-9- Hexadecenoic Palmitooleic C18:1 CH3(CH2)7CH=CH(CH2)7COOH cis-9- Octadecenoic Oleic C20:1 CH3(CH2)7CH=CH(CH2)11COOH cis-13-Docosenoic Erucic Polyunsaturated fatty acids C18:2 CH3(CH2)4(CH=CHCH2)2(CH2)6C OOH cis-cis-9,12- Octadecadienoic Linoleic C18:3 CH3CH2(CH=CHCH2)3(CH2)6CO OH all-cis-9,12,15- Octadecatrienoic Linolenic C20:4 CH3(CH2)4(CH=CHCH2)4(CH2)2C OOH all-cic-5,8,11,14- Eicosatetraenoic Arachidonic 45 Classification of Lipids Lipids Simple Complex Fats (acylglycerols) Waxes Sterides Phospholipids Glycolipids -cerebrosides Phosphoacylglycerols Sphingomyelins - gangliosides (phosphoglycerides) - cerebroside - phosphatidylcholines (lecithins) sulphatides - phosphatidylethanolamines (cephalins) - globosides - phosphatidylserines - phosphatidylinositols Sphingolipids - plasmalogens – cardiolipins 3.3 Simple Lipids Simple lipids form the alcohol and fatty acids after hydrolysis. • Acylglycerols consists of glycerol and fatty acids. Acylglycerols, namely triacylglycerols are also called neutral fats. They are the main composed part of adipocytes as molecular form of fatty acid storage. Natural tryacylglycerols are mixed lipids, because they contain different fatty acid residues. CH2 CH CH2 O O O C C C R1 R3 R2 O O O Figure 3.1 Triacylglycerol structure • Sterides are esters of cyclic alcohols (sterols) and fatty acids. Sterols are 3- hydroxyderivatives of cyclopentanperhydrophenantren. Most prevalent sterol of 46 animal origin is cholesterol, which is involved as structural lipid to the plasmic membranes structure and is precursor of vitamin D3, steroid hormones and the bile acids. HO CH3 CH3 CH H2 C CH3 CH2 CH2 CH H3C CH3 1 2 Figure 3.2 Cyclopentanperhydrophenantren (1) and cholesterol (2). • Waxes are simple lipids, which are the esters of highest fatty acids and high molecular alcohols. From waxes of animal origin there are bee wax, spermaceti, lanolin, which are used for cream production. 3.4. Complex Lipids Complex lipids have not only alcohol and fatty acids, but also additional conponent (alcohol, phosphate, nitrogen compounds, carbohydrates). Complex lipids are polar, amphypathic substances and main part of them implements structural function by involving to membrane composition. • Phospholipids. Phospholipids are divided to the glycerophospholipids and sphingophospholipids due to the alcohol, which is involved in their structure. - Glycerophospholipids are esters of glycerol and highest fatty acids. They are derivatives of phosphatidic acid esterified by amino alcohols choline, etanolamine, serine. CH2 CH CH2 O O O C C P OH O OH R1 O R2 O phosphatidic acid 47 Phosphodiesteric bond in glycerophospholipids is formed by hydroxyl group of choline (phosphatidylcholine or lecithin), ethanolamine (phosphatidylethanolamine or cephalin) or serine (phosphatidylserine). Choline 1 2 Serine 3 4 Figure 3.3 Glycerophospholipids: phosphatidylcholine (1), phosphatidylethanolamine (2), phosphatidylserine (3), phosphatidyl inositol (4). 48 In phosphatidyl inositols phosphotidic acid is linked with the six- membered cyclic alcohol inositol. Phosphatidyl inositol diphosphates are the precursors of second messengers (inositol triphosphate and diacylglycerol). Figure 3.4 Cardiolipin (diphosphatidylglycerol). - Plasmalogens differ from above phospholipids in that they, in peace of a higher fatty acid residue, contain a residue of α, β-unsaturated alcohol linked through an ether bond to the glycerol hydroxyl group at position C-1. There are three types of plasmalogens: phosphatidal cholines, phosphatidal serines, phosphatidal ethanolamines. Plasmalogens constitute 10 % of the cell membranes, they are also involved to myelin covers of nerve cells. Some of them show the strong biological activity. Much of the phospholipid in mitochondria consists of plasmalogen. Figure 3.5 Plasmalogen (phosphatidal ethanolamine.) Platelet activating factor (PAF) has been identified as 1-alkyl-2-acetyl-sn- glycerol-3-phosphocholine. It is formed by many blood cells and other tissues and aggregates platelets at concentrations as low as 10-11 mol/L. It also has hypotensive 49 and ulcerogenic properties and is involved in a variety of biologic responses including inflammation, chemotaxis, and protein phosphorylation. - Sphingophospholipids consist of one molecule of dibasic unsaturated amino alcohol sphingosine, one molecule of highest fatty acid, one molecule of nitrogen compound (most commonly, choline) and one molecule of phosphoric acid. N-acyl derivatives of sphingosine and fatty acids are called ceramides. Sphingophospholipids are phosphate esters of ceramides and choline, ethanolamine or serine. The nerve tissue is especially abundant in sphingomyelins (N-acylsphingosyl phosphocholines). Figure 3.6 Sphingomyelin. • Glycolipids are the complex esters of highest fatty acids and sphingosine and contain the carbohydrate component (namely glucose, galactose or their derivatives or oligosaccharide group). - Glycosphingolipids are combination of ceramide with one or more sugar residues. According to the carbohydrate part of molecule structure glycosphingolipids are divided to the several classes: cerebrosides, gangliosides, sufatides, globosides. a) Cerebrosides include a hexose, linked through an aster bond to the hydroxyl bond of sphingosine. Galactosyl ceramide and glucosyl ceramide are prevalent. Cerebrosides are especially abundant in the nervous all membranes (in the myelin sheath). Galactosyl ceramide is the major glycosphingolipid in brain 50 and other nervous tissue. Glucosyl ceramide is the predominant glycosphingolipid of extraneuronal tissues, but it also occurs in the brain in small amount. Figure 3.7 Galactosyl cerebroside. b) Sulfatides are sulfate derivatives of cerebrosides, the most prevalent from them is galactocerebroside sulfate. Sulfatides are found in the white substance of brain. They are acidic, negatively charged substances. c) Globosides are oligosaccharide derivatives (oligohexosides) of ceramides. Oligosaccharide residue of globosides contains most frequently galactose, glucose or N-acetylgalactosamine. Cerebrosides and globosides are neutral glycosphingolipids, because they do not contain any charged groups. d) Gangliosides are the similar to cerebrosides, but they have oligosaccharide instead of galactose residue. Oligosaccharide consists of monosaccharide (glucose, galactose) and a sialic acid, usually N-acetylnauraminic acid. The highest amount of gangliosides is observed in the membranes of ganglionar neurons. 51 •Lipoproteins are complexes of lipids with proteins. Lipoproteins are formed by non covalent bonds and various physico-chemical interactions. Lipoproteins of blood plasma are transport forms of lipids. Other lipoproteins are integral components of biological membrane. 52 Tests for Self-control 1. Which of below mentioned fatty acids are unsaturated? A. Palmitic and stearic B. Oleic and palmitic C. Linoleic and oleic D. Linoleic and arachidic E. Behenic and myrictic 2. All the below mentioned lipids contain glycerol except: A. Neutral fats B. Cardiolipins C. Plasmalogenes D. Lecithins E. Cerebrosides 3. Choose the simple lipid: A. Cerebroside B. Lecithin C. Waxe D. Kephaline E. Ganglioside 4. Lipids perform all the below mentioned functions except: A. Reserve B. Energy C. Protective D. Transport E. Structural 5. Polyunsaturated fatty acids are: A. Palmitic and stearic B. Oleic and palmitic C. Linoleic and oleic D. Linoleic and arachidic E. Linolenic and arachidonic 53 Chapter 4. Chapter 4. STRUCTURE OF NUCLEOTIDES AND NUCLEIC ACIDS STRUCTURE OF NUCLEOTIDES AND NUCLEIC ACIDS Nucleic acids – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are polynucleotides, which consist of the monomer units – mononucleotides. Nucleotides are the three-component compounds, which consist of nitrogen base, pentose residue and phosphate. 4.1 Functions of Nucleic Acids and Nucleotides • Genetic function. Nucleic acids play the main role in the conservation, transmission and realizing of hereditary information. • Cofactor function. Free nucleotides are involved to the enzyme structure as cofactors. For example: NAD, NADP, FAD, FMN take part in oxido-reductive processes; • The formation of active intermediates of carbohydrates (UDP-glucose, UDP- galactose) and lipids (CDP-choline). • Participation in detoxification processes (UDP-glucuronic acid, PAPS). • Energy function. Nucleoside triphosphates are high- energy substances, which accumulate the energy in macroergic bonds. • Modulator functions. Nucleotides can be allosteric modulators of several enzymes. • cAMP and cGMP are second messengers of hormones. 4.2 Structure of Nucleotides. Nucleotide Nucleoside Phosphate Nitrogen base Pentose Purine Pyrimidine Ribose Deoxyribose - Adenine - Thymine - Guanine - Cytosine - Uracil 54 Nucleotides consist of nucleosides and phosphates. Nucleosides are two-component molecules which contain nitrogen base and monosaccharide (pentose) molecule. Chemically nucleosides are N-glycosides of pentose and nitrogen base. N-glycoside bond is formed by N-1 of pyrimidine and C-1 of pentose in pyrimidine nucleotides and N-9 of nitrogen base and C-1 of pentose in purine nucleotides. Figure 4.1 Nucleotide structure. Phosphorylation of the certain hydroxyl groups of pentose leads to the nucleotide (nucleoside phosphate) formation. Phosphorylation can occur at fifth, third or second carbon. In the nucleic acids there are prevalently nucleoside 5`- phosphates. • Pentoses, which are involved to the nucleotide structure, are D-ribose (nucleotides, which contain ribose, are called ribonucleotides) or 2-deoxy-D-ribose (in deoxyribonucleotides). • Nitrogen bases are aromatic heterocyclic structures. According to chemical structure they are divided into two groups: purines and pyrimidines. Purine bases are adenine and guanine. Pyrimidine bases are cytosine, thymine, uracil. 55 N NN H N H2N NH NN H N O NH2 adenine guanine N N H NH2 O Cytosine H3C NH N H O O Thymine NH N H O O Uracil -O Nitrogen base O OHOH HH HH OP O- O 1` 3` 4` 5` 2` nucleoside nuclotide An important property of hydroxyl-containing nitrogen bases is their ability to exist in the two thautomeric forms – lactam and lactim depending on the medium pH. All the hydroxy- derivatives of purine and pyrimidine, constituents of natural nucleic acids, exist in a lactam form. It gives the possibility to generate intermolecular hydrogen bonds between purine and pyrimidine bases. Minor nucleotides. Besides 5 main nitrogen bases some nucleic acids contain the additional (minor) nitrogen bases in very small amounts. Minor bases are methylated derivatives of the usual nitrogen bases, for example: 1-methyladenine, 2-methyladenine, 6-dimethyladenine, 1-methylguanine, 7-methylguanine, 1- methyluracil, 5-hydroxymethyluracil, 3-methylcytosine etc. Nucleoside with unusual structure is pseudouridine, where the ribose is combined with uracil in the 5 position. The most amount of minor nucleotides is involved to the tRNA content. Human DNA contains N-methylcytosine, mRNA – N-methylderivatives of adenine and guanine. Table 4.1 The nomenclature of nucleotides and nucleosides Nitrogen base names Nucleosides Nucleotides Brief nucleotide names Full Brief RNA Purine Adenine A Adenosine Adenyl acid (adenosine-5`- monophosphate) AMP Guanine G Guanosine Guanyl acid (guanosine-5`- monophosphate) GMP Pyrimidine Cytosine C Cytidine Cytidyl acid (cytidine-5`- monophosphate) CMP Uracil U Uridine Uridyl acid (uridine-5`- monophosphate) UMP DNA Purine Adenine A Deoxy- Deoxyadenyl acid (deoxy- dAMP 56 adenosine adenosine-5`- monophosphate) Guanine G Deoxy- guanosine Deoxyguanyl acid (deoxy- guanosine-5`- monophosphate) dGMP Pyrymidine Cytosine C Deoxy- cytidine Deoxycytidyl acid (deoxy- cytidine-5`-monophosphate) dCMP Thymine T Deoxy- thymidine Deoxythymidyl acid (deoxy- thymidine-5`- monophosphate) dTMP 4.3 Structure of Nucleic Acids All nucleic acids are high-molecular polymers. The primary structure of nucleic acids is polynucleotide chain, which consists of monomers – nucleotides. The single nucleotides are combined together to polynucleotide chain by phosphodiesteric bonds, which are formed between of 3` and 5` hydroxyl groups of pentoses of neighbour nucleotides. Table 4.2 The features of nucleic acid primary structure Nucleic acids Pentoses Nitrogen bases purines pyrimidines DNA 2`-deoxyribose Adenine, guanine Cytosine, thymine RNA ribose Adenine, guanine Cytosine, uracil Nucleotide polarity. In the polynucleotide chain of DNA and RNA there are two ends: 5` end, which contains free 5` pentose hydroxyl; and 3` end which contains free 3` pentose hydroxyl. In the natural nucleic acids 5`-end (5`-hydroxyl of the ending ribose or deoxyribose) is usually phosphorylated, 3` end contains free OH group. Such nucleic acid is supposed to be polar and has the direction 5`→ 3`. DNA Structure, Properties and Functions. Biological role • Conservation of hereditary information. • Transmission of hereditary information to the descendants. The DNA duplication and transmission of the copies of parent molecule to the descendants is 57 the base of hereditary conservatism, keeping through the generations main biological signs of species. • Realizing genetic information. This biological function is existed by the transmission of information, coded in DNA, to the molecules of messenger RNA and following deciphering of this information during the protein synthesis. The complex of these actions is called the central dogma of molecular biology: DNA transcription RNA translation Protein replication Molecular mass and size of DNA molecules Molecular mass of deoxyribonucleic acids is essentially various in the different biological objects: viruses, prokaryotes, eukaryotes. • Viral and prokaryotic DNA. Viral DNA has the lowest size and molecular mass. In the prokaryotic cells the DNA amount is essentially higher than in viruses. Prokaryotic DNA is covalently closed double-chain ring. According to the increase of biological organization range the amount of nucleotide pairs in the double-chain DNA molecules increases. Prokaryotic DNA is one molecule, which is situated in the special part of cytoplasm – nucleoid. • Eukaryotic DNA. Eukaryotic DNA is situated in the nucleus and is involved to the polymorphic structure – chromatin. In the period of preparing to mitosis DNA duplication occurs and following chromatin condensation with formation of cytological structures – chromosomes. Each chromosome of eukaryotic cell contains two fully identical DNA molecules. The chromosome amount is specific for each biologic species. The molecular mass of human DNA is 1,6•1011Da, which is equal 2,4•109 nitrogen bases pairs. There is direct correlation between the increase of evolutional level, range of biological organization of living been and the amount of genetic material, which is manifested in the nucleotide pairs quantity and molecular mass of DNA. 58 Features of primary structure • Deoxyribose is involved to the nucleotide structure. • Pyrimidine nucleotides are cytosine and thymine (5-methyluracil). N N N N NH2 O HO HH HH PO O- O O A N NH2 ON O HO HH HH PO O- O C NH N N O NH2 N O H HH HH O PO O- O G NH O ON O HO HH HH PO O- O T 5` 3` Figure 4.2 The Primary structure of DNA Secondary structure All DNA molecules have the certain correlations between purine and pyrimidine nucleotides content. According to Chargaff rules: - sum of purine bases is equal to the sum of pyrimidine bases: A + G = T + C; - the amount of 6-amino groups is equal to the amount of 6-ketogroups; - adenine content is equal to the thymine content, guanine amount is equal to the cytosine amount: 59 A = T, G = C. The model of secondary structure of DNA has been proposed by J.Watson and F.Crick. The DNA molecule consists of two chains, which form right-handed helix, where both chains are coiled around the common axis. Each strand has an opposite polarity to the other. They are antiparallel. Figure 4.3 The hydrogen bonds between complementary nitrogen bases. The double chain stabilization occurs by the hydrogen bonds, which are formed between oppositely situated complementary nitrogen bases (adenine and thymine, cytosine and guanine), which explain the empiric Chargaff rules. The complementary base pairs lie inside the helix, perpendicular to the sugar- phosphate backbone, which lies outside the helix. The base pairs inside the helix are stacked one above the other. The hydrogen bonds of the base pairs and the van der Waals interactions of the stacked base pairs provides the stability of double helix. Figure 4.4 The scheme of DNA double helix (B-form). Structural features of double helix: diameter – 2,0 nm, distance between nitrogen bases along helix axis – 0,34 nm, spiral structure is repeated with 3,4 nm interval or each 10 nucleotides pairs. 60 All above mentioned properties characterize B-form of DNA. But according to the interaction with different amount of cations and water molecules, DNA forms other structural shapes: A, C or Z, which can characterize the certain physiological conditions of DNA interaction with proteins of nuclear chromatin. Figure 4.5 The molecular model of DNA (B-form). Tertiary structure In the living cell the double helix does not show the unfolding structure, but is additionally folded in the space, and forms tertiary structures – superspirals. At the superspiralized state DNA molecules in the complex with certain proteins are involved to the nuclear chromatin structure in eukaryotic cells. The supercoiled long DNA molecules form the compact structures, for example, nuclear chromosomes. Physico-chemical properties • Acid-base properties. All polynucleotides are the strong multibasic acids with low level of pK. DNA acidity is predetermined by the secondary phosphate groups, which are fully ionizated under pH < 4. Due to acidic properties and the presence on the surface of negative charges DNA molecules under physiological meaning of pH form the complexes with cations: polyamines (spermidine, spermine), alkaline proteins (histones, protamines), metals (Ca2+, Mg2+, Fe2+). • Viscosity and optical activity. High molecular mass and large length predetermine the high viscosity. The DNA viscosity depends on its conformation and essentially changes under denaturation and renaturation. 61 Due to ordered secondary structure DNA molecules are optically active and able to rotate the polarized light flatness. • Absorption in the ultraviolet region. Nitrogen compounds, which are involved to the nucleic acids structure, have the ability to absorption of the ultraviolet light at 260 nm. After polynucleotides formation the mutual influence of parallelly situated along the DNA molecules nitrogen bases pairs leads to the decrease of ultraviolet absorption. The DNA absorption at 260 nm is lower by 40% than the summary absorption of nitrogen bases - hypochromic effect. Denaturated DNA shows the higher level of absorption – hyperchromic effect. • Denaturation. DNA denaturation is the destruction of the native double helix conformation of DNA with formation of disordered single strands. Renaturation is the returning to native secondary DNA conformation. The denaturated nucleic acids lose their biological properties. The molecular basis of denaturation is destruction of hydrogen bonds between complementary nitrogen bases A-T and C-G. Denaturation of DNA is caused by: - sharp pH change to acidic or alkaline side; - heating of DNA solution. The thermal DNA denaturation is called melting. Each type of DNA is characterized by specific temperature of denaturation (Tm) due to the ratio of G-C and A-T pairs. The ratio between G-C and A-T pairs is important index of nucleotide composition of DNA molecules from different biological objects. As between guanine and cytosine three hydrogen bonds are formed the thermal dissociation of G-C pair require higher amount of energy than A-T pair, combined together by two hydrogen bonds. That’s why the melting temperature of DNA molecule is directly proportional to the G-C pairs amount in nucleic acid. 62 The Structure, Properties and Functions of RNA Ribonucleic acids are polyribonucleotides, which are divided into the main classes: messenger RNA (mRNA), transport RNA (tRNA), ribosomal RNA (rRNA). N N N N NH2 O OHO HH HH PO O- O O A N NH2 ON O OHO HH HH PO O- O C NH N N O NH2 N O OH HH HH O PO O- O G NH O ON O OHO HH HH PO O- O U 5` 3` Figure 4.6 The primary structure of RNA. • Primary structure. - ribose is involved to the nucleotide structure; - pyrimidine nucleotides are cytosine and uracil; - there are minor bases into the molecule. In contrast to DNA, RNA molecules are single chain polynucleotides (exception RNA of RNA-containing viruses). • Secondary structure of the single chain eukaryotic polyribonucleotides is characterized by the presence of parts with double helix structure. These parts of molecule are called hairpins and are formed by complementary base sequences with opposite polarity. Figure 4.7. The hairpin formation in the secondary structure of RNA 63 Such coiled regions contain 20 – 30 nucleotide pairs and alternate with unspiralized parts. The messenger RNA This RNA class constitutes 2 – 5% from common amount of cellular RNA. mRNA are the carriers of genetic information from the genome to protein- synthesizing system. Each mRNA serves as a template, which determines amino acid sequences in the polypeptide molecules, which are synthesized on ribosomes. mRNA shows metabolic unstability and highest heterogeneity of molecular mass and molecular size (from 25•103 till 1 - 2•106) with sedimentation constancy from 6 till 25 S. Messenger RNAs, particularly in eukaryotes, have some unique chemical characteristics. The 5` terminal of mRNA is “capped” by 7-methylguanosine that is linked to an adjacent 2`-O-methyl ribonucleotide at its 5-hydroxyl through the three phosphates. The cap is probably involved in the recognition of mRNA by ribosomes, and it stabilizes the mRNA by preventing the attack of 5`-exonucleases. The other end of most mRNA molecules, the 3`-hydroxyl terminal, has attached a polymer of adenylate residues 20 – 250 nucleotides in length. It maintains the intracellular stability of mRNA by preventing the attack of 3`- exonucleases. Secondary structure of mRNA is characterized a multiply intrachain double- spiral parts, which contain 40 – 50% of nucleotide composition. Transport RNA tRNA constitutes 10 – 20% of cellular RNA. Their molecules are polyribonucleotide chains, which consist of 70 – 90 nucleotides. Molecular mass is 23 – 28 kDa, sedimentation constant – 4 S. There are at least 20 types of tRNA in the cell according to the amount of natural L-amino acids, which interact with tRNA during the translation. 64 • Primary structure of tRNA contains a high amount of minor nucleotides (methylated nitrogen bases, pseudouridine and dihydrouridine residues). • Secondary structure looks like the clover leaf which is formed by the specific interaction of complementary nitrogen bases along polyribonucleotide chain. Unpaired nucleotide sequences form specific for tPNA structural elements: Figure 4.8 tRNA structure. - Acceptor arm is 3` end of molecule which contains terminal nucleotide sequence CCA. The ending adenosine accepts the amino acid by 3` hydroxyl of ribose during translation. - Dihydrouridine arm (D arm) consists of 8 – 12 nucleotides and contains 1 – 4 dihydrouridine residues. - Anticodon arm contains the nucleotide sequence to be complementary to triplet (codon) within mRNA. This loop provides the tRNA interaction with the mRNA during translation. - Extra arm. - Pseudouridine arm contains obligatory nucleotide sequence – 5`-TΨC-3`. This loop is seems to be necessary for interaction of tRNA with ribosome. • Tertiary structure. Characteristic for tRNA structure “clover leaf” can form more compact space conformation. Usually tertiary structure of tRNA looks like to Latin letter “L”. 65 Ribosomal RNA Ribosomal RNA (rRNA) is the class of cellular RNAs, which are involved to prokaryotic and eukaryotic ribosome composition. rRNAs constitute 90% of total amount of cellular RNA. Ribosomal RNAs together with specific proteins form the basis of ribosomal structure and functions. Ribosomes take part in the translation, that is biosynthesis of polypeptide chain on the template of mRNA. Ribonucleic acids of this type are metabolically stable molecules. By interaction with ribosomal proteins they perform the function of structural framework for organization of all intracellular protein synthesizing system. Minor bases amount in rRNA composition is essentially lower than in tRNA. But ribosomal RNAs are also highly methylated polyribonucleotides, where methyl groups are combined with or nitrogen bases or 2`-hydroxyl groups of ribose. The secondary structure of rRNA is characterized by a high amount of short double-spiral regions, which look like to the hairpins or sticks. Besides the above mentioned RNA classes in the mammal nuclei there are pibonucleic acids with different molecular mass, known as heterogeneous nuclear RNAs (hnRNA). Their molecular weight can be higher than 107. hnRNAs are the immediate products of gene transcription, they are processed to generate the RNA molecules. 66 Tests for Self-control 1. Purine bases are: A. Adenine, guanine B. Uracil, thymine C. Cytosine D. Pseudouridine E. All the above mentioned 2. Pyrimidine bases are: A. Adenine, guanine B. Guanine, thymine C. Uracil, tymine, cytosine D. Pseudouridine E. All the above mentioned 3. Biologic functions of DNA are: A. Preservation of hereditary information B. Transfer of genetic information to descendents C. Realization of genetic information D. Preservation and transference of information E. All the above mentioned 4. Nowadays about 50 minor bases have been found in the t-RNA structure besides the main four nitrogene bases. Choose the minor nitrogenous base: A. Cysteine B. Dihydrouracil C. Uracil D. Cytosine E. Adenine 5. Which statement about the base content of DNA is incorrect? A. A = T B. G = C C. A + T = G + C D. A + G = T + C E. All the above mentioned answers are correct 67 Chapter 5. ENZYMESChapter 5. ENZYMES 5.1 Structure and role. General principles of catalysis: ● Catalysts change the velocity of a chemical reaction and are not consumed during the reaction. ● Catalyst is removed from reaction without changing. ● Catalyst can cause the reactions which correspond to thermodynamic laws. ● Catalyst can not change the reaction direction and the state of equilibrium of reactions ● Catalyst decreases the energy of activation of reacting molecules (that is energy barrier). Figure 5.1. Energy scheme for enzymatic and nonenzymatic chemical reactions ● The concentrational effect is characteristic for catalysis. Biological catalysts are enzymes. Enzymes are specific proteins, which perform catalytic function. They have all the physico-chemical properties of proteins. Catalytic RNAs Certain ribonucleic acids (RNAs) exhibit higher substrate – specific catalytic activity. These RNAs are termed ribozymes. Although, the substrates of ribozymes are limited to the phosphodiester bonds of RNAs. Ribozymes catalyze transesterification and hydrolysis of phosphodiester bonds in RNA molecules. 68 2 nonezymatic enzymatic Free energy 2 ΔEe ΔEne S 1 1 - initial state ΔG P 2 – transition state 3 3 – final state 3 P Ribozymes play key role in the intron splicing events essential for the conversion of pre- mRNAs to mature mRNAs. Because enzymes are proteins they have specific properties. Specific properties of organic catalysts, or differences between enzymes and inorganic catalysts: ● High efficiency of action. ● High specificity of action. ● They act under physiological conditions (as so called soft conditions): 37°C - temperature of body; physiological pH; normal pressure. ●Cooperative effect. ● The oriental effect is typical for enzymes. ● They practically do not form side products. ● Enzymes are regulated. Structure of enzymes Enzymes Simple Conjugated protein nonprotein part part (apoenzyme) (cofactor) thermolabil thermostabil apoenzyme+ coenzyme holoenzyme Apoenzyme provides the specificity of enzyme (its selective interaction with substrate). Cofactor determines a type of reaction. The same cofactor may be included into the structure of different enzymes. For example: pyridoxal phosphate is cofactor both aminotransferases and decarboxylases of amino acids and other enzymes. 69 Cofactors Coenzymes Activators Coenzymes are defined as heat-stable, low-molecular weight organic compounds required for the activity of enzymes. Most coenzymes are linked to enzymes by noncovalent forces. Those which form covalent bonds to enzymes may also be termed prosthetic qroups. Activators are not included in enzymes as integral structure components. They support enzymes in catalytically active state (metal ions, reductive substances) Classification of coenzymes depending on the chemical structure: - derivatives of vitamins (TPP, pyridoxal phosphate, HSCoA, FH4); - dinucleotides (NAD, FAD); - nucleotides (UTP, ATP, CTP); - complexes of porphyrins with metal ions. Active site of enzyme is a unique combination of amino acid residues in the enzyme molecule that provides a direct interaction of enzyme with the substrate molecule and its immediate involvement in the act of catalysis. Active site Catalytic site Binding site (anchoring site) Catalytic site is directly involved in chemical interaction with the substrate and its conversion. Residues of Arg, His, Lys, Asp, Glu, Ser, Cys, Tyr are usually included in catalytic site. Anchoring site is responsible for the specific affinity of enzyme for substrate and the formation of an enzyme-substrate complex. Allosteric site is a site on the enzyme molecule that serves for binding low - molecular – weight compounds (effectors, or modifiers), whose molecules are structurally dissimilar from the substrate molecules. The attachment of an effector to allosteric site produces a change in the tertiary and quaternary structures of enzyme molecule with the resulting change of 70 active site configuration, which leads to the changing enzymatic activity. These enzymes are named allosteric enzymes. They are usually oligomeric proteins. Isoenzymes are multiple forms of an enzyme. They catalyze the same reaction, but differ from each other by the primary structure and physico–chemical properties, affinity to substrate, maximal rate of the reaction, regulatory properties.They are oligomeric molecules with dissimilar protomers. For example: Lactate dehydrogenase consists of four protomers of two types (H and M). Only the tetrameric molecule possesses catalytic activity. HHHH HHHM HHMM HMMM MMMM i1 (LDH1) i2 (LDH2) i3 (LDH3) i4 (LDH4) i5 (LDH5) In norm each tissue is characterized by specific isoenzyme spectrum. H4 form is predominant in the heart; while M4 form – in skeletal muscle and liver. These findings are widely used in the clinical practice, for differential diagnosis of organic and functional lesions of tissues and organs.