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Date Created: 02/16/16
e BILZSS Test 1 Spring 2001 P People Francois Jacob Said the aim of modern cell and molecular biology is to interpret the properties of the organism by the structure of it as constituent molecules Hooke Published Micrographia discovered Cellulae walls of cork Leeuwenhoek Publishes Animacules Used magnifying glass to see little animals in pond water F Redi Disproves Spontaneous Generation Said all life arises from pre eXisting life Schleiden 86 Schwann Said tissues are made of cells this began Cell theory there theory lead to the formal birth of Cell Biology Virchow First person to use the term Vital Units Basically cells come from other cells Walter Flemming First detailed description of chromosome cell division process including mitosis Jacques Loeb Removed sea urchin eggs and chemically induces embryonic development Wohler Was the rst to synthesize natural biological products which was urea from urine and oxalic acid from spinach Mulder Isolated a brous acid precipitate which turned out to be a protein Miescher Isolated DNA from sperm of Rhine sh Buchner Found out that yeast cell eXtracts convert Glucose into Alcohol Fischer De ned a peptide bond and also discovered 16 out of the 20 Amino acids know today Mendel Father of genetics characterized dominance recessive dihybrid crosses his true value was taking a quantitative approach towards genetics Correns Tschermak DeVires Continue Mendels work Sutton Boveri Developed Meiosis and Chromosomal theory inheritance Johnnsen Coined the term Gene Morgan Did work on Drosophilia fruit ys he determined things like linkage seX chromosomes and mapping genes Beadle Found out that Drosophilia eye mutant is due to a defective enzyme Beadle 8C Tatum Developed the One GeneOne Enzyme Theory basically one gene will transcribe only one enzyme Avery Macleod 8C McCarty They designed an experiment to tell if Protein or DNA is the genetic material Proteases did not alter transformation of bacteria but DNAases did DNAases destroy DNA while proteases Protein Thus indicating that DNA is the genetic material Alfred Hershey 86 Martha Chase Used 32P and established that DNA is the genetic material in viral infections Watson 86 Crick Discovered the structure of DNA Chargaff Found out the base pairing rules A T and G C Wilkens 86 Franklin Developed Xray diffraction Pauling Found that proteins form alpha helixes and beta sheets Sanger Discovered the molecular structure of insulin Holley Khorana 8C Nirenberg Discovered the Genetic code the codon UUU translate the amino acid phe Mitchell Discovered Chemiosmosis Sharp 8C Roberts Discovered that RNA has Introns and Exons and that introns are spliced out of RNA Cech 8C Altman Discovered the catalytic properties of ribozymes they are made of RNA Abbe Optimized the microscope design lens 8C condenser Zeiss Brought the lens resolution near limits of light Lacassagne Developed autoradiography which uses light microscopy and imagers Coons Developed fluorescent tagged antibodies Ruska Developed the 15t transmission electron microscope Palade 86 Porter Developed the electron microscope stains for ultrastructure Robertson Found the Unit membrane hypothesis Muhlethaler Develops freeze fracture electron microscope GPalade C DeDuve A Claude Got a Noble Prize for using the electron microscope to find the inner workings of cells Svedberg Invents the ultracentrifuge Beherens Use the centrifuge to isolate nuclei DeDuve Use the centrifuge to isolate lysosomes Jack Szostak Developed the replicase system novel ribozymes and deoxyzymes ssDNAs with catalytic activity Gerald Joyce 8C Martin Wright Did work on ribozymes they used a test tube of ribozymes that can reproduce indefinitely some even with mutations which improved rate of replication Payen 86 Peroz Alcohol precipitate of barley holds heat labile components convert starch to sugars Kuhn Coins term 39enzyme39 Greek quotin leavenquot Ducleaux Uses suffix quotASEquot for enzyme names TheoriesProven Ideas Cell Theory All living things are made of cells Vitalism Was school of scientific thought that attempts to explain the nature of life as resulting form a vital force Reductionism The primary methodological approach used in cell and molecular biology during the 20th century concept of trying to interpret the properties of a living organism by a detailed study of its constituent molecules or their individual properties Mechanists Believed that life is essentially a mechanical process it can be explained entirely by workings of laws of physics and chemistry without a vital force Cell characterization No unique laws just got the living state highly structures evolution metabolize self replication regulate exchange communication grow divide differentiate show animation die Theory of RelativityAlbert Einstein If speed of light is a constant then time and motion are relative to the observer and mass and energy are equivalent EMC2 Atomic Structure 8C F ission Rutherford discovered atomic particles Otto Hahn 8C Fritz Strassman split the nucleus of a atom releasing energy fission 2nd Law of Thermodynamics Is a measure of degree of order of the Universe its randomness can only increase Vocabulary Biochemistry Isolation and characterization of cell substances Cells Small membrane bounded compartments filled with concentrated aqueous solutions of reactive chemicals Cells more complex definition Self contained self assembling self adjusting self perpetuating isothermal mix of biomolecules 3 D conformation held by weak non covalent forces extracts precursors and free energy from surroundings catalyze reactions with enzymes which it makes has great efficiency and economy of metabolic regulation maintain steady state far from equilibrium self replicate suing DNA Evolution Changes in the allele frequency of a populations gene pool from one generation to another generation All organisms are believed to evolve via Natural Selection Gene Medelian unit Genetics Study of inheritance and characters in plant and animals Spontaneous Generation Life arises from non life Vital force A soul peculiar to living organisms and different from all other physical forces found outside living things Insulin Made by beta cells of islets of Langerhans protein of two chains and alpha chain 21 amino acids and a beta chain with 30 linked by sulfur atoms Chemiosmosis Cell energy transduction Escherichia coli Human colon bacteria Tensegrity view of cell structure the microtubules of the cytoskeleton might be the compression struts and the microfilaments and intermediate filaments might be the tensile wires Tissue Homogenization Plummeting tissues with a mortar and pestle later homogenizers blenders and sonicators blender but with sonic action are used Centrifugation Spinning samples very quickly you will have sedimentation Sedimentation Separating particles by size and density Biomolecules Selected for their fitness to perform certain biochemical reactions that are the characteristics which define life Autotrophs phototrophs 8C chemotrophs make foods from light and chemical Heterotrophs Consume food stuffs Food Stuffs Carbohydrates Proteins Fats Hormones Specific effect on activity of cells and remote from molecules point of origin Neurons Convey sensory information from one neuronal cell to another cell Replicase A molecular compleX which has the ability to make a copy of itself and direct other molecules to replicate themselves Procaryotic small unicellular simple metabolically no nucleus Eubacteria True bacteria what we find to day Chemitrophic Type of Eubacteria get energy from chemical fuels Autotrophic Type of Eubacteria get energy from photocatalytic mechanisms Aerobic Type of Eubacteria get energy from catabolic metabolism via transfer of electrons to 02 Anaerobic Type of Eubacteria get energy from catabolism of foods without 02 Archaebacteria original form oldest kind of cells Methanogens Type of Archarbacteria converts CO2 H29CH4 Halophiles Type of Archarbacteria live in the Dead Sea and Great Salt Lakes a high salt environment Thermophiles Type of Archarbacteria lives in hot springs and deep ocean geysers Eucaryotic Many internal membrane bounded organelles has a nucleus has extensive internal membranes gene in chromosomes compartmentation size 2 20 pm diameter Organelle Subcell part with a distinct metabolic function Metazoan Eucaryotic heterotrophic feeder Metaphytian Eucaryotic autotrophic producer Viruses Obligatory intracellular parasites pathogens made of protein capsid and genetic material Viron A virus particle outside the host Viroid RNA pathogen a virus wo capsid of 240 600 nucleotides Prions Proteinaceous infections particle prion protein gene encodes a protein whose accumulation leads to a degenerative disease of the central nervous system Functional Groups Groups of atoms acting as a unit that give organic molecules their physical properties chemical reactivity and solubility in aqueous solutions Most functional groups posses electronegative atoms like N P O S Some key bonds in functional groups are ester C O C and amide C N functional groups are ionizable are physiological pH Sugars Compounds with formula CH20n glucose mono di tri polysaccharides and the polymers of monosaccharides Fatty Acids Lipids Triglycerides animal fats and phospholipids membranes Amino Acids Hundreds known but only 20 are common in cells proteins Amino Acid Structure Amino acids have a carboxyl group COOH 8C amino group NH2 bound to a asymmetric carbon There are 20 amino acids and have a tetrahedron shape Zwitterion An ampholyte contains 2 groups of opposite sign Nucleotides Nitrogen containing ring compounds called N bases linked to 5 carbon sugars ribose 8C deoxyribose and a phosphate Pyrimidines Cytosine C Thyamine T Uracil U Purines Adenine A Guanine G Con guration Spatial arrangement of atoms in a molecule configuration can not be interconverted without breaking bonds isomers are based upon covalent configuration Conformation precise shape Surface outline or contour 3 D orientation of groups that are free to assume different positions in space without breakin any bonds Enzymes Can distinguish between biologically active forms based on Shape Non Covalent electrostatic interactions Individually weak but collectively strong Ionic Bonds Charged small ions which attract wo water very strong atoms gain lose ions Dipoles weak molecular force which is due to a neutral but asymmetrical internal distribution of charge within a molecule that can result in an attraction to the opposite charge Dispersion van der Waal F orces Electrostatic attraction based upon closeness of atoms involved in macromolecule interaction s for shape HydrophobicHydrophilic Interactions Repulsion of electrostatic dipoles of water by non polars favors fatty hydrocarbons groups self assembling substances that dissolve readily in water ions 86 polars water P surround and solubilize Hydrogen bond O H or N H associated with a lone pair on a N or O Covalent Bond Sharing of outer orbital electrons between 2 atoms which completes outer shells of each atom and forms a molecule Enantiomers Nonsuperimposable mirror images Isomers with identical chemical properties but rotate the plan of polarized light at different angles Isomers Compounds that have the same formula but a different arrangement of their constituent atoms Levorotatory Polarized light bent right Clockwise Dextrorotatory Polarized light bent left Counter Clockwise Metabolism Catalytic reactions of enzymes in cells Catabolism Cell respiration of heterotrophs they oxidize food stuffs the steps are 1 digestion of polymers foods 2 Glycolysis gt AcoA splitting of sugar 3 oxidation of AcoA gt CO2 NADH gt H2O ADP P gt ATP Anabolism Biosynthesis coupled reaction energetically unflavored with favored Free Energy AG AH TAS AG is a numerical measure of how far a reaction is from equilibrium Coupled Reactions linking hydrolysis of ATP favored to thermodynamically unfavored reactions creating biological order Autotrophs Light energy covalent chemical bond energy Heterotrophs Food stuffs OxidationReduction Redox Reactions e H are transferred between oxidized 8C reduced forms Oxidation Removal of e from a substrate Reduction Gaining of e 8C often a proton as well H Peptide bond Formed by condensation reaction between amino of one amino acid 8C carboxyl of another amino acid Protein Polymer of L amino acids joined by peptide bonds Insulin 2 polypeptides control carbohydrate metabolism alpha chain of 30 amino acids 86 beta chain of 21 amino acids Glucagon Pancreatic hormone 29 amino acids opposes insulin action Corticotropin 39amino acids anterior pituitary hormone that stimulates adrenal cortex Oxytocin 9 amino acids hormone of posterior pituitary that stimulates uterine contractions Bradykinin 9 amino acids hormone stimulates smooth muscle vasodilation 8C inflammation responses Angiotensin Octapeptide derived from angiotensinogen by kidney enzyme renin increases blood pressure Thyrotropin Releasing factor TSH 3 amino acids of hypothalamus stimulates thyroid release of thyroid hormone Enkephalins Either of two pentapeptides with opiate and analgesic activity occur naturally in brain 8C have marked N Aaffinity for opiate receptors compare endorphin Enzymes Responsible for catalytic activity 86 function Transport Proteins Bind 8C carry ligands Storage Proteins EX ovalbumin gluten 8C casein ferretin Contractile Motor Can contract change shape elements of cytoskeleton actin myosin tubulin Structural support Collagen of tendons 8C cartilage elastin of ligaments keratin of hair feathers 8C nails fibroin of silk 8C webs Defensive protect Antibodies IgG fibrinogen 8C thrombin snake venoms bacterial toxins Regulatory signal Regulate metabolic processes hormones transcription factors 86 enhancers growth factor proteins Receptors detect stimuli Light 8C rhodopsin membrane receptor proteins and acetylcholine or insulin Simple Proteins Yields only amino acids on hydrolysis Albumins Soluble in water globular mostly enzymes Globulins Soluble in dilute aqueous solutions insoluble in pure distilled water Prolamines Insoluble in water soluble in 50 to 90 simple alcohols Glutelins Insoluble in most solvents soluble in dilute acids bases Protamines Not based upon solubility small MW proteins with 80 Arginine 86 no Cysteine Histones Unique structural complexed with DNA high content basic amino acids 90 Arg Lys or His Scleroproteins Insoluble in most solvents fibrous structure cartilage 8C connective tissue Collagen Type of Scleroproteins has high Glycine Proline 86 no Cysteine when boiled makes gelatin Keratins Type of Scleroproteins proteins of skin 86 hair it has a high amount of basic amino acids Arg His Lys but with Cys Lipoproteins Type of complex proteins found in blood membrane 8C transport proteins Glycoproteins Type of complex proteins found in antibodies cell surface proteins Nucleoproteins Type of complex Proteins found in ribosomes 8C organelles Primary Protein Structure Sequence of amino acids Secondary Protein Structure Regular recurring orientation of amino acid in a peptide chain due to H bond Tertiary Protein Structure Complete 3 D shape of a peptide Quaternary Protein Structure Spatial relationships between different polypeptides or subunits Polymorphism Proteins may vary in primary sequence but have same function Polymorphism inter specific Between species diff amino acids sequences Polymorphism intra specific Within a species liver vs kidney Invariants Don39t vary significantly in amino acids sequence examples ubiquitin mito 8C histones chromosomes Site Specificity Sequences determine intra cell location signal sequences prosthetic binding sites Families of proteins Different but related functions evolved from a single ancestral protein 30 commonality of sequence serine proteases trypsin chymotrypsin elastase Homologous Proteins Evolved in related fashion diff species but perform same cellular function in diff species ex cytochrome C in duck 8C chickens 2 variants in yeast 8C horses 48 variants Functional Groups OH Hydroxyl Alcohol NH2 Amine Amino Acid COOH Carboxyl Acid CH3 Methyl Hydrocarbon CO Carbonyl AldehydeKetone SH Sulfhydryl Disulfide PO4 Phosphoryl Phosphate C CO C Ester C CONH2 Amide CH3 Methyl Hydrophobic Types of Reactions Functional group transfers Glu ATP lt gt G6P ADP Redox reaction oxidation reduction PGAld lt gt 13di PGA Rearrangement isomerizations G6P lt gt Fruc6P C C breaking or re formation F 1 6bP lt gt DHAP 3PGAld Condensations Proteinn aa gt Proteinn1 Hydrolysis Glu Glun H20 gt Glu Glun 1 Classes of amino acids classi ed by R Groups Acidic Negatively charged ASP 85 GLU R group with 2nd COOH that ionizes above pH 70 Basic Positively charged LYS ARG HIS R group with 2nd amide that protonates below pH 70 Polar Uncharged SER THR TYR ASN GLN Are soluble in water ie hydrophilic Non Polar aliphatic GLY ALA VAL LEU ILE PRO Contain only hydrocarbons R groups hydrophobicity Aromatic Hydrophobic nonpolars PHEMET T RPCYS All contain R groups with ring structures Peptide Bond Characteristics Partial double bond character Shorter 8C stronger than C C Longer yet weaker than CC No free rotation group in same plane TRANS results is zig zag planar molecule Protein shape 85 Conformation Native Protein Conformation Is a 3 D spatial shape that39s most thermodynamically stable it has the lowest free energy expenditure 8C forms spontaneously Helix A spiral staircase like shape Fiber Elongated bound monomers Globular Roughly a sphere Physical forces Non covalent bonds H bonds hydrophobic 86 hydrophilic interactions 86 covalent bonds as peptide bonds 86 disulfide bonds Native Conformation of most enzyme proteins is globular Interior is pocket of hydrophobic exterior is hydrophilic maximizes H bonds that form Chaperones proteins that bind to others 86 help facilitate native folding in most energetically favorable way Protein Secondary Structure Characteristics Alpha Helix Peptide backbone around long axis core rigid cylinder R groups radiate outward 36 amino acid per turn Single repeat turn of helix 360 o 054 nm Right handed helix counterclockwise Helix formed from Hbond interactions H of N of any amino acid 8C OC of 4th amino acid 14 of amino acids in globular proteins occur in alpha helix Beta sheet A linear extended zig zag pleated sheet formed by H bonds intra 8C inter chain Domains Structural units within larger peptides made of helix 8C sheets allowing maximal H bonds Motifs Combos of domains repeated in many different 8C unrelated proteins Hairpin Beta Motif 2 antiparallel beat sheets joined by a sharp turn loop Beta alpha beta motif 2 parallel beta sheets connected by a helix 4 helix Bundle 4 06 helicies connected by 3 bends form a pocket for CoE 86 metal ion ligand binding sites ex cytochrome B562 and TMV coat protein 06B Saddle Sheets fold to make a core that looks like a saddle ex LDH BB Sandwich Criss cross patchwork of b b sheets that form a hydrophobic pocket ex insecticyanin antitrypsin BB Barrel 8 sheets occur in a circle each connected by helix link ex PYR kinase glyaldehyde P isomerase RuBP carboxylase immunoglobulin antibody Protein Tertiary Structure Characteristics 3D orientation of proteins in space Thermodynamically most stable conformation Weak non covalent interactions 8C S S bridges Hydrophobic interior 86 hydrophilic exterior Myoglobin MW 16700 Cytochrome C MW 12400 heme binding single polypeptide of 100 amino acids in ETS of mitochondria Lysozyme MW 14600 enzyme egg white 86 human tears 124 aa39s with 4 S S that hydrolyses poly saccharides in bacterial cell walls bactericidal agent Ribonuclease MW 13700 enzyme of 124 amino acid w 4 S S Denaturation Loss of 3 D conformation by heat pH organic solvents detergents Renaturation Regaining of biological activity Protein Quarternary Structure Characteristics 3 D conformations between more than one polypeptide or subunit of a protein ex hemoglobin RNA polymerase ASP transcarbamylase pyruvate dehydrogenase Some Common Quarternary Level Protein Shapes Dimers self recognizing symmetrical regions which bind together identical binding sites Tetramers 4 identical subunits F ilaments polymers of subunits each bound together in an identical way forming a ring or helix see Colied coil 2 parallel helicies forming a stiff filament with a stripe of hydrophobic amino acids see Molecular Techniques Crude Cellular Homogenates Use mortar 8C pestel or tissue grinders or blender or sonicators with a buffered medium with substrate to rupture cells making homogenate Differential 8C Ultra Centrifugation Produces a pellet solid 86 supernatant liquid repeated centrifugations at increasingly higher speeds separates organelles by their mass and density Velocity Sedimentation Seperation accourding to size in a gradient of sucrose between 510 while under a centrifuge you will get sedimentation the smaller particle in the bottom and larger closer to the top Equilibrium Centrifugation By bouyant densitydensity gradient centrifugation in 30 to 70 sucrose or CsCl cell part move up or down into bands independent of size 86 shape where their density equals the density of tubes sucrose CsCl Partition Chromatography Small molecular weight molecules partitioned between phases of 2 solvents water alcohol Paper Chromatography Uses cellulose Thin Layer Chromatography Silica gel on glass plates Column Chromatography In cylindrical glass column on permeable support media retards flow of selected molecules other pass through Ion exchange Chromatography Charged ligands matrix retards passing proteins of opposite charge DEAE cellulose dimethylaminoethyl cellulose CM cellulose carboxymethyl cellulose Gel Filtration Size exclusion chromatography inert matrix retards smaller size proteins Sephadex Af nity Chromatography Based on biological activity an inert polymer with ligand antibody enzyme subst binds a specific protein High Pressure Liquid Chromatography HPLC Sample is vaporized and injected moves through a column containing stationary liquid phase under high pressure separate into compounds according to their affinity for the stationary phase Gel Electrophoresis Porous gel starchpolyacrylamide separation is by size 86 charge SDS Electrophoresis SDS PAGE Detergent SDS binds 1 SDS 2 amino acids proportional to MW Isoelectric Focusing A pH gradient in a glass column of gel proteins move to point of its pI ie no charge 2 Dimensional Electrophoresis Combines isoelectric focusing with SDS electrophesis if gel is turned at right angel 86 SDS PAGE is done Auto radiography Cell biology technique in which radioactive precursor molecules are localized within microscopic thin sections of cell tissues by photographic film emulsion exposure Peptide Map protein ngerprint Treat a puri ed protein with proteolytic enzyme Distinctive fragments electrophoresed or chromatographed Spectroscopy Spectroscopy Measures intensity of light beam before 86 after it passes through a sample comparing two intensities over a range of wavelengths Percent transmittance Ratio of intensity of light passing through the sample to the intensity of light shining on sample multiplied by 100 Absorbance is the log of the transmittance absorbance at 280 nm measures aromatic aa39s Colorimetry colored dye binds to amino acids Ninhydrin reaction rx39s w amino blue color 10 9 M Biuret test mg quantities based on Copper ion binds stiochiometrically violet color Bradford test ug amounts based on dye Coomassie blue binds peptide Fluoroescamine dye pg quantities10 12 M Quanti cation of protein present is based on BEER LAMBERT Law linear relationship between light Absorbance vs Concentration aminginto 1 55 1quot 1E mi mgmms grownu 1 international unit of Enzyme Activity Amount of protein which converts 1 micromole of substrate to product per min at 25 O C at optimal pH eX Urease 1 unit will liberate 10 mmole of ammonia from urea per minute at pH 70 at 25 C equivalent to 10 IU 1 unit Speci c Activity Units of enzyme activity per mg protein 1 unit Molecular Activity Number of units of enzyme activity per mole of enzyme Enzyme Regulate metabolic reaction rates control metabolism molecules mostly protein that accelerate or catalyze chemical reactions A gtB in cells by breaking old covalent bonds and forming new covalent bonds a biological catalyst but different from a chemical catalyst have compleX structure sequence of amino acids act only upon a speci c substratedo not change direction energetics of reactions Reaction Path E S lt gt ES lt gt E P Enzymes Catalyze Reactions By lowering the energy of activation Isoenzymes isozymes Enzymes that have the same catalytic function but have a different chemical structure primary sequence Substrate What an enzyme acts upon Product The substance left after an enzyme acts on a substrate Cofactor Small ions mostly metal ions Cu Mg Mn act as activators 8C inhibitors Coenzymes Small non protein ligands catalyze reactions electrons transfer a group break form a bond Lipoic acid Oxidative de COOH alpha keto acid NAD NADP Dehydrogenation H carrier and or electron transfer CoASH Acyl carrier via sulfhydryl SH Vitamins acscorbate cyanocobalamin folic acidetc Prosthetic Group Large complex organic molecules which may have catalytic activity heme Active site Portion of enzyme which precisely fits the contours of a substrate by weak electrostatic interactions Mechanism of Action The chemical reaction scheme by which an enzyme acts upon a substrate Lysozyme Active site is a long groove holding siX sugar units has 2 acidic side side chains ASP 8C GLU this enzyme cuts polysaccharide by hydrolysis adds H 2 O breaks glycosidic bond C O C via distortion EC MAJOR CLASSES Oxidoreductases dehydrogenases Catalyze oxidation reduction reactions often using coenzyme as NAD FAD Alcohol dehydrogenase ethanol NAD gt acetaldehyde NADH Transferases Catalyze the transfer of functional groups Hexokinase D glu ATP gt D glu 6 P ADP Hydrolyases Catalyzes hydrolytic reactions adds water across C C bonds Carboxypeptidase A aa aan H 2 O gt aa aa n 1 aa Lyases Add or remove groups to CC bonds Pyruvate decarboxylase pyruvate gt acetaldehyde CO 2 Isomerases mutases Catalyze isomerizations Maleate isomerase maleate gt fumarate Ligases Condensation of 2 substrates with splitting of ATP Pyruvate Carboxylase PYR CO 2 ATP gt 0AA ADP P Leonor Michaelis 85 Maude Menten Kinetics Proposed mathematical modeling of enzyme reactions an algebraic expression of rectangular hyperbola k 1 k 3 ESlt gtESlt gtEP k 2 k 4 Assumptions 1 Rate formation ES complex from E P is negligible ie can ignore the rate constant k 4 2 Rate limiting step is disassociation of ES to E P k 3 3 Important state of the enzyme is termed Free Enzyme Free enzyme E t ES Bound enzyme ES Total enzyme E t E ES ES Km the Michaelis Constant is a mathematical interpretation of an enzyme action is substrate concentration at which rate is equal to Vmax is a characteristic physical property for each different enzyme is independent of E if there39s more than 1 substrate then each has its own Km measures quotRELATIVE a initjl of an enzyme for its substrate one enzyme with 2 substrates with following Km39s 01 M 86 005 M one takes more substrate to reach the same Vmax many enzymes have individual steps in a complex reaction sequence each with their own Km not all enzymes are treatable by M 8c M kinetics most regulatory enzymes multi subunits are not treatable Enzyme Inhibition Irreversible inhibitor molecule can not be easily removed from enzyme ie enzyme is physically altered by binding of inhibitor Alkylating agents like iodoacetamide bind to SH s Organophosphorous compounds nerve gases SER Reversible Enzyme activity may be restored by removing the inhibitor and are thus treatable by M 8c M kinetics 2 major types of reversible inhibitions Competitive Inhibitor binds to E forms an EI complex at the active site Inhibitor often looks like substrate fools active site 86 binds Extent of inhibition is concentration dependent ie can be overcome if S is very high ie S gtgtgt I Classical example is malonic acid inhibition of SDH Easy to demonstrate is via Lineweaver Burke plots Shows VmaX is SAME 8C Km is increased Non Competitive Inhibitor binds to E forms an EI complex not at active site Inhibitor bears no structural relationship to substrate Removes a net amount of active enzyme ie lowers total E Can NOT be overcome even if S is very high Easy to demonstrate via Lineweaver Burke plots Shows Km is SAME 86 VmaX is different Enzyme Regulation 3 methods 1 By of enzyme molecules present gene action 2 By sequestering compartmentalizing lysosomes 3 By adjusting reaction rates of eXisting enzyme Stiochiometric Controls amount substrate present Feedback Inhibition negative regulation An initial enzyme is inhibited by end product Allosteric Regulation modulation negative or positive Protein eXists in multiple forms Active Form of Enzyme A conformation favoring activity Inactive Formof Enzyme Non favorable Allosteric proteins have 2 binding sites Active site for substrate and Allosteric site binds regulator Phosphorylation Changes protein conformations Reversible Protein Phosphorylation Transfers Phosphate done by protein kinases which transfer Phosphate from ATP Protein Phosphatases Dephosphorylate GTP Binding Proteins G Proteins Active when GTP is bound to protein Inactive when the GTP is hydrolyzed to GDP serve as molecular switches esp cell signaling LG later Primary Mechanism of Phosphorylation Substrate Level Phosphorylation Chemiosomosis Oxidative Phosphorylation subst H NAD gt NADH subst NADH gt H proton motive force a ATP Photosynthetic Phosphorylation light NADP gt NADPH gt H Cell Respiration Series cytoplasmic 8C mitochondrial linked enzymatic pathways stepwise oxidation of food molecules makes ATP Physiological view uptake of O 2 86 release of CO 2 Biochemical view 02 consumption CO2 production 3 Stages 1 Digestion food polymers gt monomers 2 Production ofAcoA gt glycolysis 8C Fonidation 3 Oxidation ofAcoA to CO2 86 H2 0 gt KC 86 ETC Cellular Pathways Glycolysis Glucose gt pyruvate NADH ATP Kreb39s Cycle AcoA gt CO2 NADH GTP FADH2 Electron Transport Chain ETC Passage of e39s from NADH to 02 gt H2 0 H gradient ATP synthase mitochondrial membrane protein which makes ATP as H move into mitoplasm Glycolysis Anaerobic no requirement of oxygen Cytoplasmic location 10 step enzymatic pathway Hexose gt 2 PYR 4ATP 2 net 2NADH Energy investment phase coupled Rx39s Phosphorylation of low energy intermediates Energy capture phase g 45p116 Redox reaction glyceraldehydeB PDH Substrate level phosphorylation Glycolysis and Ancillary Pathways Fates of Pyruvate If anaerobic 1 alcoholic fermentation via alcohol dehydrogenase 2 lactic acid respiration LDH If aerobic Krebs Cycle Shuttles purpose to move e39s from cytoplasmic NADH to mitochondrial NADH or FADH2 Glycerol P shuttle skeletal muscle brain Malate shuttle liver kidney heart muscle Key Reactions of Glycolysis Substrtae level phosphorylation Redox reaction involving NAD Summary of Glycolysis 2 ATP used to initiate pathway 4 substrate level phosphorylations makes 2 ATP net 2 NADH and 2 Pyruavte Fermentations 8C Shuttles Krebs Cycle Krebs Cycle Citric Acid Cycle Tricarboxylic Acid Cycle A cyclical biochemical pathway resulting in aerobic oxidation of cell fuels as CH2 0 fatty acids 86 amino acids while making CO2 H2 0 86 ATP Overall reaction Acetyl CoA 3NAD E FAD GDP P 2H2 O gt CoASH 3NADH E FADH2 GTP 2CO2 Enzymes of Krebs Cycle 5 Dehydrogenases ISDH a aKGDH SDH MDH 8C PDH 2 Hydrolyases aconitatse 8C fumarase 1 Thiokinase succinyl thiokinase 1 Synthetase citrate synthatase 2 multi enzyme complexes each with 60 proteins 86 5 coenzymes 1 Pyruvate Dehydrogenase 2 Alpha ketoglutarate dehydrogenase Key Metabolic Reactions of Krebs Cycle NAD is reduced Substrate level phosphorylation occurs Decarboxylation COOH Acylation Via CoASH Each turn of the cycle 4 protons passed to coe39s 3NADH 8C 1 FADH2 2 CO239s are released 3 parts of Mitochondrial Oxidation of Pyruvate 1 PYR gt C02 H2 0 gt NADHFADH2 Krebs 2 e ofNADHFADH2 gt 02 to make H2 0 ETS 3 ADP P gt ATP Chemiosmosis Pyruvate Dehydrogenase Complex Oxidative decarboxylation of alpha Keto acid Pyruvate Acetyl Co A HOOC C CH3 gt CoA S C CH3 CO2 II H O O 3 Enzymes a Pyruvate Decarboxylase 12 dimers 24 identical subunits b Dihydrolipoyl Transacetylase reductase 8 triamers 24 identical subunits each 3 lipoates c Dihydrolipoyl Dehydrogenase 6 dimers 12 subunits with FAD 5 Coenzymes Vitamin 1 CoASH Pantothenate 2 Lipoate Lipoic acid 3 Thiamine pyrophosphate Thiamin B1 4 E FAD Ribo avin B2 5 NAD Niacin Regulation of Krebs Cycle Controls ow of intermediates in 8C out Substrate availability mass action Allosteric inhibition end productfeedback inhibition Covalent modification reversible phosphorylation Protein kinases 8C phosphoprotein phosphatases 4 key enzymes are involved in regulation PDH Citrate Synthetase Isocitrate dehydrogenase Alpha keto gluatarate dehydrogenase Fatty Acid Metabolism beta oxidation Oxidation of Fatty Acids to Acetyl CoA 3 Steps in Fat Oxidation Cycle 1 Oxidation of COOH end of free fatty acid 2 Transport of fatty acyl coA into mitoplasm 3 Oxidation of 2 carbon fragments as AcoA 4 enzymes of beta oxidation 1 Fatty acyl coA ligase on outer mito membranes FA COOH ATP CoASH lt gt FAcoA AMP PP converts cyotplasmic FA to F atty acyl coA 2 Carnitine acyl transferase 1 outer mito memb F attyCoA carnitine lt gt Fatty acyl carnitine CoASH transfers FAcoA to carnitine for transport across mitochondria 3 Carnitine acyl tranferase 2 in mitoplasm Fatty acyl carnitine CoASH lt gt FAcoA carnitine releases FAcoA inside the mitoplasm 4 Fatty acyl coA dehydrogenase Beta Oxidation Cycle Four steps for these dehydrogenase enzymes a dehydrogenation with FAD gt FADH2 b hydration addition of water c dehydration W NAD gt NADH d thiol clevage W CoASH releases a 2c piece AcoA Net result each turn of the cycle shortens a long chain fatty acid by 2 carbons generating 1 AcoA 1 NADH and 1 FADH2 Mitochondrial Membrane Transport 8C Electron Transfer Membrane impermeant to most everything esp to H outer membrane porins molecules 5000 10000d inner membrane 70 protein 8C 30 lipid contains a Redox proteins of ETC b ATP synthase c Carrier proteins phosphate translocases ADPATP translocases pyruvateH symporter d Glycerol P 8C malate shuttles Mitochondrial DNA 16 500 np39s codes for 20 mito proteins subunits of NADH dehydrogenase and 22 tRNA39s and 2 rRNA39s How Electron Transfer Works Redox Potential empirical measure of tendency to gain e39s strong reducing agent has negative 0 Eo39 strong oxidizing agent has positive 0 Eo A G039 nf A Eo NADH ltgt NAD H 2e 032V H20 lt gt 12 02 2H 2e 082V AGo39 10023 114 262 Kcal Electron Transfer Chain39s Order gt aligned linearly by increasing Redox Potential from electronegative to electropositive to and therefore by energy differentials Components of the ETC Pyridine nucleotides NAD enzyme bound hydrogen carriers accept 2e39s and or protons Flavoproteins FMN 8C FAD protein bound hydrogen carriers Iron sulfur proteins FeS non heme iron electron carriers Ubiquinone CoQ semiquinone 8C hydroquinone mobile membrane bound non protein hydrogen carriers Cytochromes a a3 b562 b566 Cl C quotcolored proteinsquot with bound Fe atoms ferrric vs ferrous iron porphyrin heme bound protein carriers Mitochondrial Respiratory Assemblies NADH Q reductase Succinate dehydrogenase Cytochrome C Reductase Cytochrome Oxidase II39WEEI39EE 39iE il E E miE Ii l l WEEI39IDFEII39IE I39Il 39Ei H Emil iE m 39lnil1nrrln 1quot HalEH n H n t Emma In rm EEWWE bailti rams1h was 39 2H 4 1399 g nchmm l 39 mam ATP Synthase IIhydrophilic channel for H ow makes 100 ATP per 300 H per sec condenses ADP Pi gt ATP F0 membrane piece 8C stalk F1 soluble piece 5 proteins Oxidative Phosphorylation Making ATP Synthesis of ATP made via a proton motive gradient generated by transfer of e39s to reduce 02 86 make H20 through series of redox proteins Mechanism Chemiosmotic Coupling Mitchell 1961 a fundamental mechanism arose early in evolution 8C was retained 3 steps ETC passes e thru membrane carrier proteins PMF Proton Motive Force gradient pH difference DpH 10 units ph 80 matrix vs pH 70 peri mito space membrane otential difference D charge 140mV in vs out ATP Synthase links ADP to P making ATP uncouplers as DNP destroy H gradient no ATP Photosynthesis Light Driven Phosphorylation Production of ATP via photo phosphorylation Cellular process Bacteria Blue green and Eucaryotic cells with chloroplasts Capture of light energy by pigments chlorophylls 8C accessory pigments Capture electrons as reducing power in NADPH Reduction of CO 2 to CH 20 2 Fundamental Reaction Mechanisms Light Reactions photochemical reactions molecular excitation chlorophylls by light charge separation generation of proton motive force H gradient reduction of NADP to NADPH Via an ETS Dark Reactions thermo chemical reactions CO 2 xation reduction stages carboxylation CO2 RuBP gt 2 PGA reduction PGA NADPH gt PGAL regeneration of RuBP via HMP path gt RuBP 6C0 2 12H 20 gt C 61 1120 6 6H 20 60 2 Pigments Accessory Pigments Carotenoids carotenes xanthophylls Phycobilins chromophore a protein Phycoerythrin 8C phycocyanin Chlorophylls abcd etc side chain differences Absorption Spectra Plot of the amount of light absorbed by a solution vs the wavelength of light Action Spectra Plot of physiological activity eg O 2 evolution APs vs the wavelength of light Molecular Excitation of Chlorophyll Absorption of Light Energy blue light 440nm 715 Kceinstein red light 700nm 409 Kceinstein Ground State paired e39s with opposite spin stability absorption moves non bounded e39s to higher orbitals lst excited singlet state 2nd excited singlet state 1st long lived state Fates of Absorbed Light Energy 1 Re radiated as vibrational heat 2 Re radiated as uorescence emission of light of longer wavelength 700 nm gt 710nm in time frame 10 9sec less energetic 3 Re radiated as phosphorescence emission of light much longer wavelength 700nm gt 720nm in real time lsec 4 Induced resonance vibrational e excitation inducing like vibrations in adjacent molecules causing their excitation 5 Photoionization enters into the photochmical reactions loses electron to acceptor ionized chlorophylls Photosynthetic Electron Flow Photosystem chlorophylls reaction center primary acceptor PS 1 and PS 2 path of e flow cyclic vs noncyclic release of O2 capture of e into coenzyme NADP gt NADPH ATPase makes ATP just like in mitochondria chemiosmosis 85 its location in chloroplasts Dark Reactions of Photosynthesis occur in stroma chloroplasm consume ATP and NADPH made in light reactions reduces xes CO2 into CHZO sugars 3 different pathways to make sugar 1 C3 CALVIN cycle 1 CO2 5C RuBP gt 2 3C sugars PGA 2 3C sugars combine gt 1 net glucose RuBP carboxylase 50 of leaf protein Photo respiration inhibition by O 2 2 C4 Hatch 8C Slack pathway 1 CO 2 3C PGA gt 4C acid mesophyll cells 4C acid gt BC CO 2in bundle sheath CO 2 into Calvin cycle as above 3 CAM Pathway C4 same as C4 but in same cell temporal not spatial differences Morphological Basis of Photosynthesis Plastids Double unit membrane bound organelles classi ed by pigment content functional Proplastids Progenitor plastid found in MERISTEMATIC cells 1 to 3 umeter dia often 20 cell spherical to ellipsoid few primary thylakoids do hold lipid droplets divide by flssion like process Leucoplasts Defined via function Amyloplasts synthsize 86 store starch Aleuoplasts contain stored protein crystals Elaioplasts contain oil 86 fat globules fat biosynthesis Chromoplasts Found in ower petals ripe fruit scenescent leaves end point of plastids differentiation formed from leucoplasts 8C chloroplasts not proplastids contain numerous water soluble anthocyanin pigments of unknow function Differentiation Elaboration of membrane enzyme systems 8C enlargement Proplastids gt chloroplasts leucoplasts chromoplasts Chloroplasts gt chloroplasts 86 or chromoplasts Leucoplasts gt chloroplasts 86 or chromoplasts Chloroplast Ubiquitious to all green plants defined by its containing chlorophyll Shape Variable elipsoid ovoid lenticular stellate convex Size 2 to 3 um dia by 5 to 10 um long Number 15 to 20 perr mesophyll cell 400000 cc Volume Often much larger than mitochondria Chloroplasm Stroma Pyrenoids Which are starch coated protein granules 70s Procaryotic Ribosomes Naked DNA 2 to 10 fg DNAchlp equals bacterial cell DNA highly supercoiled 8C repetitive 6 copies Enzymes of C02 xation and lipid droplets Thylakoid Membranes