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THE NUCLEIC ACIDS 1 Friedrich Miescher in 1871 Isolated what he called nuclein from the nuclei of pus cells Contain phosphates Nuclein was shown to have acidic properties, 1889 Altman called it as nucleic acid © 2007 Paul Billiet ODWS 2 Watson & Crick (1953) Structural model for DNA (Nobel prize 1962) 3 STRUCTURE OF NUCLEIC ACID Nucleoproteins H2O NA + Protein H2O Nucleotides Nucleosides + H3 PO4 2 purine bases + 2pyrimidine bases + A pentose sugar © 2007 Paul Billiet ODWS 4 Purine base or Pyrimidine base Pentose Sugar 5 Nucleosides Two components 1. Pentose Sugar - Ribose or 2-Deoxyribose 6 Nucleosides contd… 2. Heterocyclic Nitrogenous base Substituted Purine or Pyrimidine 7 Purine nucleosides Base O H sugar Adenine H Nucleoside β - Glycosidic link Adenosine 2-deoxyadenosine 8 Base O sugar 4 3 2 Pyrimidine nucleosides 5 2 6 1 4 3 5 4 2 1 6 1 H Thymine H 5 4 3 3 6 2 1 5 6 β - Glycosidic link 9 Ribose – thymine & 2-deoxy-uracil not found among the hydrolytic products of natural NA 10 Nucleotides 1. 2. 3. Three components Pentose sugar Heterocyclic Nitrogenous base Phosphate group 11 Base Phosphate H3PO4 + P O O sugar Nucleoside sugar Base Nucleotide Nucleotides are phosphate esters of nucleosides 12 O HO P Phosphoric acid OH OH O HO - H2O Adenosine P OH Adenosine monophosphate (a ribonucleotide) 13 O O HO P HO P OH OH OH 4 Thymidine 2 5 Thymidine monophoshate (a deoxyribonuxleotide) 14 15 Nucleic acids Very high molecular weight polynucleotides Important biopolymers of cells Responsible for biosynthesis of proteins and transmission of hereditary character. 16 Nucleic acids P P G G P P C C-5’ – C-3’ P C Phosphodiester linkage P C C P P A A P P T T P P T Nucleotides Biological properties are determined by the sequence of bases T Polynucleotide or Nucleic acid 17 Two types Ribonucleic acid (RNA) Deoxyribonucleic acid (DNA) Sugar unit – Ribose Sugar unit – Deoxyribose Nitrogenous bases – Adenine, Guanine (purines) + Cytosine, Uracil (Pyrimidines) Nitrogenous bases – Adenine, Guanine (purines) + Cytosine, thymine (Pyrimidines) 18 Structure of RNA Single stranded Some portions aquire double helical pattern A-U G-C 19 20 Types of RNA 1. mRNA or template RNA Single stranded Some degree of coiling: no base pairing Most stable & heterogeneous in size Mol mass: 30000-50000 Free state in cyttoplasm or associated with ribosomes 5% of total cellular RNA Carries genetic information from chromosomal DNA to ribosomes for protein synthesis 21 22 2. tRNA or soluble RNA Single stranded Bending and looping to clover leaf pattern Smallest polymeric form of RNA Mol mass: 25000 70-100 neucleotide units Occurs in cytoplasm 15% of total cellular RNA Carry specific AA from AA pool of cytoplasm to ribosome 23 3. rRNA Single stranded High degree of coiling; double helical regions Highest mol mass: 1.2x 106 Most abundant (80%) Present in ribosome Provide correct orientation to mRNA 24 Structure of DNA Sugar- deoxy ribose Bases: A, G …..purine T, C ….. Pyrimidine Two polynucleotide chains: Double helical A :T… 2 H bonds G:C…..3 H bonds 25 Two strands: complementary but not identical A T G C T T A C T A C G AA T G Diameter 20Ao Avg distance between two adj. base pairs= 3.4 Ao 10 base pairs / turn 26 Biological functions 1. Replication 2. Protein synthesis 27 Biological functions 1. Replication DNA Contain genetic information as specific base sequence 28 Protein synthesis Genetic code: Genetic information for protein synthesis : sequences of bases in DNA Message in A G C T language Message Read, translated & expressed Two steps Transcription Translation 29 1. Transcription Transcribing genetic information from DNA to mRNA Process • Unwind DNA helix • One acts as template for synthesis of mRNA • Build-up of complementary nucleotides along template DNA strand : enzyme RNA polymerase • According to Base pairing principle DNA : A C G T mRNA: U G C A 30 • DNA return to its original double helical structure mRNA diffuses from nucleus to cytoplasm 31 2. Translation Transferring the transcribed information from mRNA to polypeptide chain to get specific protein Process Cytoplasm : mRNA attached to rRNA –protein combination and genetic code deciphered Triplet of base along mRNA codes particular AA: codon Eg: GGA : glycine All 20 AA have triplet code Transcribed code in mRNA read by tRNA 32 tRNA looped structure On one of its loops it carries a triplet of bases; anticodon One of its trailing end AA ( AA pool)is attached through high energy ester bond catalyzed by aminoacyl-tRNA synthetase tRNA transport AA to the correct site of mRNA on ribosome particles 33 tRNA Structure 34 tRNA line up on mRNA through H bonding Each codon on mRNA is matched by its complementary tRNA anticodon Eg: mRNA codon: GGC tRNA anticodon: C C G glycine AA linked through peptide bonds form polypeptide chain (protein) catalyst: peptide synthetase Protein released from ribosome 35 36 37 Genetic Code AA sequence of proteins is predetermined in DNA which is transcribed to mRNA Genetic Code: The nucleotide base sequence of DNA that specifies the AA sequence of protein 4 bases of mRNA code for all 20 AA in polypeptide chains of various If one base for one AA; 41 = 4: only 4 AA can be specified 38 2 base for 1 AA; 42 = 16 doublet codes; 16 AAs can be specified 3 base for 1 AA; 43= 64 triplet codes; more than sufficient for 20 AAs Only 20 AAs but 64 triplet codes More than one code for some AAs Some combinations are not code for AAs 39 Codon: Each triplet of bases strung consecutively along mRNA molecule that codes for particular AA Codon Sense codon Non-sense codon Non-sense codon: stop or termination codon: signals the termination of protein synthesis 40 64 Codon 61Sense codon 3 ( UAA, UAG, UGA) Tryptophan (UGG) & methionone (AUG) : 1 codon each Others : many codons Sense codon which code for same AA: degenerate or synonymous codon Eg: GGU, GGC, GGA, GGG code glycine 41 Mutation Alteration in the sequence of nucleotide bases of DNA molecule Can ocuurs spontaneously or brought about by external agents Lead to abnormal changes in DNA replication & the defect may pass along to the next generation as an inherited factor 42 Mutation (contd…) Lead to production of protein with altered AA sequence: affect biological activities and lead to abnormalities and deseases Possibility for the development of cancerous cells Causes: some chemicals Hydroxylamine NH2OH high energy radiations (x-rays, gamma rays) Disrupt some bonds in DNA molecule and will re-form in another sequence\ 43 Thymine dimerisation 44 28-10-2013 45 Enzymes Biological catalysts Reactions are successful, efficient and high rate 1860: Louis Pasteur detected enzymatic activity in living cells- ferments 1878: Friedrich Kuhne introduce the term Enzyme = ‘in yeast’ 1897; Edward Buchner extracted enzyme from yeast cells 1926: James B. Summer isolated an enzyme in pure crystalline form (urease) 46 Enzymes > Biological catalyst > Catalyze biochemical reactions in the cells > Capable of acting independently of the cells > Highly specialized class of proteins N H2CONH2 + H2O …….> 2NH3 + CO2 (urease) C12H22O11 + H2O ……> 2 C6H12 O6 ( maltase) 47 Biochemistry : enzymology Living cells require thousands of different enzymes to catalyse the metabolism of carbohydrates, fats, proteins, etc Endoenzymes: eg:- Respiratory enzymes Exoenzymes: eg:- digestive enzymes 48 Chemical nature of enzymes Chemically high molecular mass globular proteins Simple protein enzyme: molecular structure are made up of α- aminoacid units only Eg:- pepsin, trypsin amylase, etc Conjugated protein enzyme: contain a non-protein part with protein part Protein part: Apoenzyme Non-protein part: co-factor Apoenzyme + co-factor = holoenzyme 49 Chemical nature of enzymes Neither cofactor nor apoenzymes can be active Only combination can show catalytic activity Cofactor: bridge between Apoenzyme and substrate If cofactor firmly bound to apoenzyme: Prosthetic group Eg: Fe-porphyrin combination in cytochrome Fe – heme combonation Cofactor : inorganic moeity(Mg2+, Ca2+, Zn2+.etc) : activator or organic moeity ( B- vitamin)- coenzyme 50 General characteristics A. Functional 1. Efficient: Increase the speed up to 10 million times that of uncatalyzed reactions 2. Unalterability 3. Small quantity 4. Speed up the attainment of equilibrium, but not alter the position 51 5. Specificity: Choosy about substrate Eg: Urease: hydrolysis of urea Alcohol dehydrogenase: dehydrogenation of primary alcohols 52 B. Condition characteristics (Factors influencing enzyme action) 1. pH: optimum pH- at which catalytic efficiency will become maximum for most enzymes optimum pH around 7 gastric enzyme pH around 2 2. Temperature: Optimum temp.: maximum catalytic activity Animal enzyme: 35- 45oC Plant: 40- 60oC High temp. activity lost due to denaturation 53 3. Susceptibility to the action of enzyme regulators Regulators Inhibitors Activators Increase the catalytic activity of enzymes Eg: Zn 2+ : alcoholic dehydrogenase Mn 2+ or Co 2+ : arginase Decrease or destroy the catalytic activity of enzymes Eg: Ag+,, Hg2+, Pb2+,etc: urease 54 4. Dependence on substrate concentration . © 2007 Paul Billiet ODWS 55 Theory of enzyme catalysis 1. Michaelis-Menten Theory 56 Koshland’s Induced fit hypothesis Active site: flexible and elastic Substrate induces some configurational changes in active site New configuration perfectly matching with that of substance The active site is reverts to its original configuration after the product is detached from the enzyme 57 Koshland’s Induced fit hypothesis 58 PART IV CELLULAR ENERGETICS & METABOLISM 59 Biochemical reactions Anabolism: Simple molecules + Energy .…> macromolecules Catabolism Macromolecules ……> simple molecules + energy Anabolism + catabolism = metabolism 60 CELLULAR ENERGETICS: Thermodynamics Gibbs- Helmoltz eqn: ∆G = ∆ H -T ∆ S Feasible (spontaneous) : ∆G = -ve ∆G = -ve : exergonic reactions (Catabolism) Eg: step wise degradation of sugar … CO2 + H2O ∆G = +ve : endergonic reactions (anabolism) Eg: amino acids ….> peptides 61 Coupled reactions If ∆ G +ve , coupled with reactions having large & -ve ∆ G so that net ∆ G –ve : coupled reactions Coupling is a fundamental mechanism of bioenergetics All energy requiring processes proceed by coupling A ……> B ; ∆ G > 0 (endoenergetic) ----- (1) P……> R ; ∆ G < 0 (exoenergetic) ------(2) (1) + (2) P R+E ∆ G = (∆ G1 +∆ G2) < 0 A+E B 62 Not direct Two taking place at different time & at different place in the cell Link between exo- and endo-energetic reactions Adenosine TriPhosphate Energy released first used to generate ATP ATP on hydrolysis ADP + Energy 63 Store and transport the energy of catabolic reactions Anabolic reactions taking place at proper site, time and rate ATP: Nucleotide 64 Oxidation Food ADP + P ATP ADP E Products + E ATP ADP + P ΔG = -31 kJ/mol AMP + P ΔG = -28.5 kJ/mol 65 Not a long term storage form of energy Consumed at a high rate : stock in the cell is very small As it is being used up, it has to be replenished: need energy Phototrophs (algae, plants, some bacteria) use solar energy: photosynthesis Chemotrophs ( eg; S-bacteria, nitrifying bacteria) use chemical energy from oxidation of inorganic compounds heterotrophs (humans animals, etc) consume biomolecules produced by photoptrophs 66 Biological role Reservoir of chemical bond energy Link between endergonic & exergonic reactions hydrolysis ATP ADP + Energy(cell functions: muscle movement, uptake of nutrients, etc) Indispensible for the transport of substances across the cell membrane Energy source of all cellular reactions: universal energy currency of the cell 67 Metabolism o o o o o o molecular processes by which living system acquire and utilize energy needed for life processes Aggregate of biosynthetic and biodegradative processes Anabolism Catabolism Coupling reaction ATP 68 Energy need for the synthesis of ATP Catabolism of carbohydrates, lipids and proteins Before synthesis of ATP, nutroients have to be digested and absorbed into body/ body fluid Digestion: breakdown of ingested complex foodstuff into simple molecules by hydrolysis\ Small molecules can be absorbed through the walls of alimentary canal into body luids and used for metabolism Carbohydrate: simple sugars Lipids: fatty acids Proteins:AA 69 METABOLISM OF LIPIDS Digestion …..> fatty acids Metabolism of lipidS = catabolism of fatty acids First stage: β-oxidation fatty acid cycle (spiral) Catalyzed by a set of enzymes : fatty acid oxidases 1st stage : fatty acid cycle’ 2nd stage: Krebs cycle 70 Fatty acid cycle (spiral) 71 NAD+ and FAD : oxidizing agents, reduced to NADH & FADH2 Under cytochrome series NADH & FADH2 and FAD + ATP: electron transport chain Most of the associated energy is released & stored in this stage of lipid metabolism β-oxidation + Krebs cycle + electron transport chain) NAD+ (stearic acid ) 147 ATP molecules 45% stored energy + 55% dissipated as heat 72 METABOLISM OF PROTEINS 1. 2. Proteins:AA 1st step: elimination of amino group: α-keto acid Two group transfer reactions Oxidative deamination Transamination 73 Oxidative deamination AA is converted to keto acid by NAD+ or FAD: amino acid oxidases NH2 …..> NH3 …….> Urea (excreted) Significant for Glutamic acid: α-ketoglutaric acid is an intermediate in Krebs cycle 74 75 Transamination AA and α-keto acid mutually exchange their NH2 and CO groups Catalyzed by transaminases having coenzyme; pyridoxal phospahte 76 77 Pyruvic acid converted into acetyl coenzymeA, then degraded through Krebs cycle Aspartic acid …….> oxaloacetic acid COOH- CHNH2- CH2-COOH …..> COOH – CO CH2-COOH All AA can make their way to the Krebs cycle 2nd stage: Krebs cycle and electron transport chain Deaminated moeity oxidised to CO2 + H2O + ATP 78 Wilkins & Franklin (1952): X-ray crystallography © Norman Collection on the History of Molecular Biology in Novato, CA 79 80