301-2.pptx elememtry biochemistry introduction

Biochrmistry

1. Biochem-301, 3(2-1) new Elementary Biochemistry By Dr. Zahid Mushtaq

2. Theory A general introduction to the science of biochemistry. Ionization of water, weak acid and weak bases, pH, buffers, diffusion, osmosis and osmotic pressure. Enzymes: Classification, nomenclature, characteristics, coenzymes, cofactors and prosthetic groups. Mechanism of enzyme action. Enzyme inhibition. Carbohydrates: Classification, characteristics, aerobic and anaerobic oxidation of glucose, biological functions of carbohydrates. Lipids: Composition and classification, structures of saturated and unsaturated fatty acids and their properties, characteristics of fats and oils, general metabolism of fats and oils. Proteins: Composition and classification, characteristics and classification of amino acids, peptides and levels of structural organization of proteins, physiological function and general metabolism of proteins. Nucleic acids: Chemical composition, structures of DNA and RNA. Functions of DNA and different types of RNA in the cell .

3. Introduction to Biochemistry Chemical Structures, reactions, principles and mechanism behind such interactions with all aspects around  Chemistry Biomolecule’s -Chemical Structures, reactions (Metabolism) , principles and mechanism behind such interactions with life in all its deverse forms and aspects either directly or indirectly  BioChemistry Molecular Level “Molecular logic of Life in all its diverse forms can be explained by Biochemistry”

4. Deals with metabolic processes in living tissues  a material called Protoplasm (basis of all life) These reactions in Normal way  HEALTHY But Disorganization SICKNESS/ DEATH All components that make life are themselves inanimate but combinations makes life possible. Young emerging science in the 20 th century but now a major Discipline  dependent on the discoveries of braches of Chemistry (organic, inorganic, physical , analytical etc), Physiology

5. Scope and importance of Biochemistry Now it answers to explanations for the mechanisms behind Medical sciences  Physiological, Pharmacology, Bacteriology, Pathology, nutrition, food sciences etc Solutions to clinical problems, remedies to deficiencies like Rickets , pellegra (B3), Beri-Beri (B1 Thiamin), Scruvy , Anemia Diagnosis and therapy Purifying vitamins, hormones (insulin), Anti-toxins, vaccines, proteins etc Enzyme inhibitors (Drugs e.g competitive), Recombinant DNA technology/ genetic engineering , cloning , DNA profiling (identification) Mysteries in agriculture, industry, research and all life sciences

6. Books recommended Principles of biochemistry by Lehninger (4 th edition and onward) http://www.irb.hr/users/precali/Znanost.o.Moru/Biokemija/Literatura/Lehninger%20Principles%20of%20Biochemistry,%20Fourth%20Edition%20-%20David%20L.%20Nelson,%20Michael%20M.%20Cox.pdf Medical biochemistry by Mushtaq Ahmed vol-1 edition after 2008 Cell and molecular biology by Gerald Karp 3 rd edition and onward any http://www.btsdl.cc/cell-and-molecular-biology-by-gerald-karp-6th-edition-tf2432083.html

7. THE CELL CELL THEORY; All living organisms are made up of cells and cellular components. May be uni /multi cellulars . Basic structural and functional unit of Life All cells are produced from preexisting cells. Properties ; A high degree of chemical complexity and microscopic organization . Systems for extracting, transforming, and using energy from the environment to do work Defined functions for each of an organism‘s components and regulated interactions Mechanisms for sensing and responding to alterations in their surroundings. A capacity for precise self-replication and self-assembly. A capacity to change over time by gradual evolution.

8. Plasma membranes and cytoplasm “The outer periphery of the cell that separates its internal contents from the surroundings.” Lipid and protein molecules Thin, tough, pliable, hydrophobic barrier Selectively permeable by Transport proteins Signals by receptor proteins Membrane enzymes participate in reactions Flexible in shape and functions  less strong bondings Can grow as cells multiply Cytoplasm: “The internal volume enclosed by the plasma membrane” Aqueous portion cytosol and particular portions gel Rich in enzymes, RNA, metabolites, macromolecules, coenzymes , Ribosomes and Proteasomes

9. Movement of materials across membranes Cells surrounded by plasma membrane  all communications through it Dual function of membrane 1) must retain the contents avoid leakage 2) exchange of necessary materials Lipid bilayer is best to protect loss of ions, polar, amino acids, sugars, hormones etc Movements are  passively (gradients, no energy ) and actively (energy needed)maintains net flux (one may exceed other)

10. Passive transport: Simple diffusion, diffusion through aqueous protein-linked channel, facilitated diffusion Spontaneous process in which substances move from a region of higher concentration to low concentration till equilibrium Exergonic  energy from external source for random thermal motion / collisions If substance Electrolyte  movement by chemical gradient or by Electric Potential gradient i.e. by concentrations or by difference between charges electrochemical gradients E.g. K + ions across membrane ++++++++++++++++++++++ 3Na - - - - - - - - - - - - - - - - - - 2K/ Cl -

11. Channels  Move ions  nerve impulses, secretions, muscles contractions, cell volume, open stomata etc Integral membrane proteins Downhill Always bidirectional till net flux Sequence similarities shows this protein has common ancestry Keeps open/close conformations Voltage gated channels: Conformational changes depends on difference of ionic charges on 2 sides. E.g. K+ channels Ligand-gated: Conformational changes depends on binding of a specific ( ligand ) molecule which is not solute itself. E.g. acetylcholine binds to outer surface to cation channels, cAMP binds inner to Ca++ channel. C. Elegans (1000 cells), 90 different genes K + channels

12. Non-electrolyte diffuses passively… Substance must be in high conc at one of membrane Membrane  Permeable to it Solute must cross aqueous pore without contact with lipid Solute must cross lipid layer by dissolving It must have polarity match (NON-POLAR) Partition coefficient: Ratio of solubility of solute in non-polar solvent ( octanol /veg.oil) to that in water, when both solvents are mixed together 2 molecules  same P.C, then small uncharged will penetrate faster. E.g. more CO2,O2,H2O, NO etc & Less sugars, amino acids, P-compounds require mechanisms P.C Pentration cm/sec sugars caffein Small values less permeability Greater lipid solubility greater penetration

13. Diffusion of water through membrane best movement example Water usually moves rapidly as compared to other ions / polar solutes  selectively permeable  membrane  called OSMOSIS “Water moves readily across a semi-permeable from a region of lower solute concentration to a region of higher solute concentration” Demonstrated by placing a cell in a non-permeable solute solution of different concentrations across plasma membranes Hypertonic / hyperosmotic solution Cells shrink and own H2O comes out  cured by gain of ions Hypotonic / hyposmotic soltn Cells swell by gaining H2O from outside  cured by loss of ions Isotonic 0.85% NaCl saline No net flux Temporary but best example of movement and shape changes

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15. Uses Digestive tract secrete  Liters of of fluid but reabsorbed by osmosis Animals remain in iso -osmotic , but in diahhrea occurs if fails to reabsorb Plant cells hypertonic internally  let H 2 O inside Turgid (turgor pressure pushes against cell walls) in hypertonic solutions plasmolysis Aqua porins in some cells more permeable to water kidney/plant root protein-channels allows this In congenital nephrogenic diabetes insipidus  mutations in aquaporins  vassopressins fail no reabsorbtion  higher urine excretions

16. Facilitative diffusions: (fast diffusions by selectively binding membrane spanning proteins that helps in diffusion) Solute binds selectively at one end induces Conformational changes exposing it to other end. This transporter binds at a side with solute at one time then to the other side Similar to enzyme- catalysed reactions; Specific to even D/L isomers Obey saturation kinetics i.e. blocked at saturation Regulated Conformational changes are induced (induce fit model) Slow rate 100s to 1000s /sec For Example: GLUT1-5 isoforms , insulin responsive cells in muscles and adipocytes . High glucose insulin GLUT4 more translocated uptake into cells

17. Properties and functions of water Polar Molecule of H 2 O 70% by weight Liquid state due to H-bonds Between others Between each other Like dissolves like , so polar solutes and solvents dissolves by interrupting H 2 0-H 2 O interactions Higher melting, boiling points and heat of vaporization, great internal cohesion are all due to H-bonds . Hydrophilics those that dissolve easily Hydrophobics & Amphipathics

18. AS SOLVENT

19. Ionization of water, weak acids and bases Proton hoping Water is weakly ionizable i.e. few molecules dissociate although its neutral . Many properties can also be explained by this. No free proton exists in water but forms hydronium Ionization  Electrical Conductivity and proton hoping makes it fast (cathode/anode movements)

20. To express ionization quantitatively For a reversible reaction we know; Equilibrium constant K eq = fixed and characteristic for any given chemical reaction at a specified temperature. It defines the composition of the final equilibrium mixture For water; At 25 o C [H2O]=55.5M, for 1L water its 1000/18=55.5 Ionic product of water E.C=1 x 10 -16 M = K eq

21. At neutral pH we have same concentrations of both H+ & OH- Thus when H+ is high OH- should be Low. Vice versa Similarly we also know that; [H+] [OH-] = 1 x 10 -14 M 2 Taking –log on Both sides ; –log[H+] –log [OH-] = –log [1 x 10 -14 M 2 ] Since we know –log [H+]= pH Thus pH + pOH = 14 At neutral pH, [H + ]= 1 x 10 -7 M

22. pH, pOH , pKa pH= - log [H+] where [] represents molar concentration Strength of H+ in a solution that indicates the measure of acidic/basic character. Since addition of acids and bases changes ions concentration in indexes or powers of 10 and in decimals. Logarithm changes it into an expressible whole number forms. i.e. 1 x 10 -7 =[H+]= pH=7.0=neutrality pOH = -log [OH-] pKa = - log Ka ( defined as that value of pH at which the amount of weak acid and its conjugate base are equal ) Its helpful in determining the strength of a buffer as pKa±1=buffer capacity , Ka is the dissociation constant of an acid. More values stronger acids highly ionizable.

23. The pH of an aqueous solution can be approximately measured with various indicator dyes, including litmus, phenolphthalein, and phenol red, which undergo color changes as a proton dissociates from the dye molecule. Accurate determinations of pH in the chemical or glass electrode method. pH is important in clinical sciences  acidosis/alkalosis.

24. Buffers “Are solutions or systems that tends to maintain their own pH when a small amount of acid or base is added to them” Chemically two types ; Acidic = weak acids + conjugate base/salt For Example, CH 3 COOH/CH 3 COO- or CH 3 COONa  acetate buffer or sodium acetate buffer Basic = weak base or its salts E.G., NH 4 OH / NH 4 Cl Hemoglobin is also a buffering system.

25. Buffers serve as first line of defense against any foreign invader, enzyme reactions, cell biology, storage, switch on off by maintaining structure-function relationships, microbiology etc. Buffer strength is determined by the Molar concentrations of the components making it (0.5 M + 0.5 M= 1.0 M) pKa = 4.76 , pH=4.8 (max capacity around) of acetate buffer. Among both components weak acids play a significant role in determining capacity and properties of a Buffer. pKa ±1 = Buffer Capacity Buffers…….

26. ` E.g #2: Sodium Phosphate buffers H3PO4/NaH 2 PO 4 / Na 2 HPO 4 / Na 3 PO 4 CH 3 COOH + NaOH  CH 3 COONa + H 2 O CH 3 COONa + HCl  CH 3 COOH + NaCl Common ion effect suppressed the dissociation of acid base which change pH, neutral salt / water, weak acids as products causing little changes in pH. Titration with 0.1M CH3COOH 10mL with 0.1M NaOH  to understand buffering action of weak acid Buffers……. Machanism

27. Preparation of Buffers Prepare a PO 4 buffer of pH=7.0 of 250 mL volume with 0.1 M concentration? Information is required about Potassium and Sodium phosphate salts or acid that has pKa values closest to the required pH. Information of molar ratios of weak acid and conjugate is required , from Handerson-hasselbalch equation. Molar masses required

28. Selection ; H3PO4= pKa =2.1 NaH2PO4= pKa = 6.8 Na2HPO4= pKa =12.2 pH = pKa + log [A-]/[HA] 7.0 = 6.8 + log [A-]/[HA] 0.2 = log [A-]/[HA] 0.2/1 = log [A-]/[HA] Taking anti-log on both sides to eliminate log Anti-log (0.2) = [A-]/[HA] 1.585 / 1 = [A-]/[HA] Thus molar ratio of salt (Na2HPO4) is 1.585 and acid is (NaH2PO4) = 1 Total ratios = 1.585 + 1 = 2.585 Volumes required ; Vol of Na2HPO4 = 1.585 / 2.585 x 250 mL = 153.3 mL Vol of NaH2PO4 = 1 / 2.585 x 250 mL = 96.7 mL Check ; 153.3 mL + 96.7 mL = 250 mL

29. To get information of mass or weigh of the salt and acids for making buffer; We need molar mass of Na2HPO4 . 2 H2O = 178 g NaH2PO4 . 2 H2O = 156 g Amount of Na2HPO4 . 2 H2O = 178 g x 153 mL x 0.1 M 1000 = 2.72 g in total 250ml of salt in buffer solution (dH2O) Amount of NaH2PO4 . 2 H2O = 156 g x 96.7 mL x 0.1 M 1000 = 1.508 g in total 250ml of acid in buffer solution (dH2O) Thus first take 200 mL of dH2O and dissolve Na2HPO4 . 2 H2O = 2.72 g and NaH2PO4 . 2 H2O = 1.508 g and make volume upto 250 mL Check pH Check % error Amount (g)= Mol wt x Molarity x Vol 1000

30. Problem # 2. Prepare a buffer solution of Na-Acetate of pH=5.76 , 0.1M of 1 litre volume. ( pKa =4.76 of CH3COOH)

31. Solution pH = pKa + log [A-]/[HA] 5.76 = 4.76 + log [A-]/[HA] 1 = log [A-]/[HA] Taking anti-log on both sides to eliminate log 10/1 = [A-]/[HA] Total molar ratios = 10 + 1 = 11 Volume of acid = 1/11 x 1000 mL = 90.9 mL Volume of salt = 10/11 x 1000 mL = 909.1 mL Selection= CH3COOH/ CH3COONa CH3COOH specific gravity 1.052 g/ mL , 99% Amount of CH3COOH = 60 g x 90.9 x 0.1 M 1000 = 5.45 g in total 1000ml of salt in buffer solution (dH2O) Amount of CH3COONa= 82 g x 909.1 x 0.1 M 1000 = 7.5 g in total 1000 ml of acid in buffer solution (dH2O) NOTE= FOR SOLID SALT CH3COONa WE CAN WEIGH BY WEIGHING BALANCE BUT FOR LIQUIDS LIKE CH3COOH WE NEED TO USE VOLUMES EQUIVALENT TO MASS CALCULATED (Info about purity and sp.gravity will help here)

32. We know CH3COOH specific gravity= 1.052 g/ mL , 99% that is 1.052 g = 1 mL 1 g = 1/1.052 5.45g = 1/1.052 x 5.45 = 5.18 mL so 99% pure acid required in vol = 5.18 mL 1% = 5.18/99 100% = 5.18/99 x 100 = 5.3 mL of 99% pure is required to make a buffer check pH , calculate % Error, check individually while adding components which component plays a vital role in pH.

33. Numerical assignment (hand written) Calculate the concentration of H+ ions in a solution of pH=0.5 ? Calculate the OH- ions concentration in a given solution of pH 1.1? Calculate the number of molecules of glucose present in 360 grams? Calculate the number molecules of H2SO4 in 1 mL if its of 1.84 specific gravity? What will be the molar volumes of NaH2PO4 and Na2HPO4 required to make a buffer of 0.5M , if their amounts are 1.25 g and 1 g respectively used for preparing buffer. Calculate the amounts of acetic acid and sodium acetate, to prepare pH=7 buffer, of 0.25M and 175mL volume?pKa of acetic acid= 4.76, 96% purity of acid. What will be the pH of a solution containing OH- ions 1.34 x 10 -4 ? What will be the Ka of an acid with pKa value close to 4.8?

34. What are enzymes ?  Biological catalyst Enzymos  some catalytic agent derived from Yeast Catalyst is an agent that accelerates rate of the reaction V o by lowering the E a (energy of activation) without itself appearing in the products. E a  the energy in Cals/mol required to be supplied to the reaction to initiate. Both V o and E a are inversely proportional to each other. Moles of product formed per unit time is rate of the reaction Specificity  Absolute (1 En 1 S )and relative (1 En (range of substrates)  (gluco/ hexokinases ) Mode of actions  lock and key mechanism & induced fit model

35. Factors affecting enzyme’s activity or V o 1. Enzyme concentration : [E] α rate of reaction V o 1st order reaction  with increase of one reacting species or factor there is proportional increase or decrease in rate of the reaction Depending on hormones, metabolites, other factors  increase or decrease [E] i.e. rate of synthesis and degradation (in hours/days)

36. Factors 2. Substrate concentration: those following Machelis-menton kinetics  start [S] increases the Vo proportionally 1 st order reaction when substrate is less and before Km value.

37. Effect of temperature: within limited range i.e. due to denaturation of proteins of enzymes, temperature increase  increases Vo (Q 10 principle, 30 o C valid) Since temperature α K.E α V o  collisions of reactants But maximum activity seen at each enzymes’ optimum temperature. Plants tolerate max at 60 o C and humans 37 o C Effects of pH: within limited range of pH since pH changes the ionic states of proteins which keep them intact so certain ionic state of enzyme protein  structure + function (active site) But maximum activity seen at each enzyme’s optimum pH. Trypsin = 8-9 pH, salivary amylase= 6.4-6.9 Effects of products : A+B  C+D (if reversible) Reaction may proceed more faster if products removed or reactant increased Reaction rate may slow if products has similarities with substrate Cofactors and inhibitors: cofactors (bridge S  active site ) and coenymes are required to make enzyme complete and to increase rate of the reaction e.g. Fe++ ( cytochrome oxidase , catalase etc), Cu++ ( cytochrome oxidase ), Zn++ (alc. Dehydrogenase , carbonic anhydrase ), Mg++ ( hexokinase , pyruvate kinases etc), Mn ++ ( arginase ), K+ ( pyruvate kinases ), Ni+ ( urease ) Organic or metalloorganic (group transferring)  NAD, FAD, FMN, CoASH , etc coenzymes Apoenzyme (protein part)  cofactors (tightly bound, prosthetic groups) holoenzymes Inhibitors interfere

38. CLASSIFICATION OF ENZYMES OTHLIL

39. Classification HEXOKINASES = 2.7.1.1 (ATP; D-Glucose-6-Phosphate Transferases “ Ase ” after substrate  Lipase, urease , proteases Machanism = transmethylase , oxidases etc Trivial names = No relationship but may be latin etc. Pepsin, trypsin , chymotrypsin IUBMB 4-digits + systemic name + units i.u .= 1 umole [S] [P]/min at 30 o C at opt pH. Katal = mole of substrate without 30 o C More than 2 names = succinyl-coA synthatase OR succinate thiokinases

40. Types of inhibitions of Enzymes Reversible inhibitions IRReversible inhibitions IAA Thiols DIPF  Acetylcholine esterases Heavy metals (Ag+) EnZ -SH ( Mercaptides ) Ampicillin  transpeptidases (C.W)

41. Competitive enzyme inhibitions When resemblance in structure between the [I] and [S] Binds to some or all free enzymes Km and Vmax changes Lowers the [ES] complex Can be reversed by increasing [S] Important as metabolic antagonists , chemotherapy against bacterial, viral and malignant cancer cells For example; Succinate dehydrogenase tRNA ( puromycin ), glutamine( azaserine ), Met ( ethionine )

42. In cell wall synthesis and DNA synthesis and stops cells from dividing

43. No resemblance with substrate Inhibitor doesn’t binds the active site but sites other than active site. Binds to both [E] and [ES] Causes change in the 3-D structure of active sites No effect of increasing [S] For examples ; Ions of heavy metals EDTA chelates Ca ++ Fluoride removes Mg ++

44. Binds to only [ES] complex. Makes [ESI] complexes Not reversed by adding excess [S] but may be by changes like pH, temperature, etc For example; Where two or more substrates are required like DNA polymerases being inhibited by ddNTPs

45. Isozymes / iso -enzymes Enzymes that catalyze same reaction (same organism) but are Physically & Chemically distinct as produced at different locations. Lactate dehydrogenase (LDH I-V) Creatine Kinase (CK 1-3) Each 34,000 Mol. Wt on electrophoresis

46. Chemically distinct LDH 2 genes seperately encoding 2 polypeptides ( H & M types) Combines into a 4-polypeptides unit LDH-1= H4 (heart), LDH-5=M4 (skeletal muscles) CK/CPK 2 polypeptides  B & M Combines into Dimer CK-1= BB (Brain), CK-2=MB (Heart myocardium), CK-3= MM LDH-2 & CK-2= Myocardial Infarction in serum appears (DIAGNOSIS) Lungs, kidney, heart ,liver etc Brain, gonads, heart ,retina, muscles etc

47. Ribozymes “Certain RNA molecules have catalytic properties similar to Enzyme therefore called ribozymes” Substrates Others RNAs or part of ribozymes itself Therefore believed to be first gene first enzyme evolutionary. As in HIV viruses reverse transcriptases cDNA

48. Examples of ribozymes 414 bp RNA in venomes of tetra himena catalyses self elongation from host nucleotides M1-RNA in Rnase-P precursor t-RNA cleavge RNA in self splicing of spliceosome mRNA Small RNA viruses of plants RNA part of rRNA in Ribosome  peptidyltransferase activity Iron- protoporphyrin -rings  heme  Catalase H2O2H2O + O2 Benzidine test ( Blood detection )

49. Proteins and Amino acids