BIL255 Exam 1 Spring 2001 Definitions

BIL255 Exam 1 Spring 2001 Definitions - e BIL255 Test#1...

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Unformatted text preview: e BIL255 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 & 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 first to synthesize natural biological products, which was urea from urine and oxalic acid from spinach. Mulder- Isolated a fibrous acid precipitate, which turned out to be a protein Miescher- Isolated DNA from sperm of Rhine fish Buchner- Found out that yeast cell extracts convert Glucose into Alcohol Fischer- Defined 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 flys) he determined things like linkage, sex chromosomes, and mapping genes Beadle- Found out that Drosophilia eye mutant is due to a defective enzyme Beadle & Tatum- Developed the One Gene=One Enzyme Theory, basically one gene will transcribe only one enzyme. Avery, Macleod & 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. 32 Alfred Hershey & Martha Chase- Used P and established that DNA is the genetic material in viral infections. Watson & Crick- Discovered the structure of DNA Chargaff- Found out the base pairing rules A-T, and G-C Wilkens & Franklin- Developed Xray diffraction Pauling- Found that proteins form alpha helixes and beta sheets Sanger- Discovered the molecular structure of insulin Holley, Khorana, & Nirenberg- Discovered the Genetic code, the codon UUU translate the amino acid phe Mitchell- Discovered Chemiosmosis Sharp & Roberts- Discovered that RNA has Introns and Exons and that introns are spliced out of RNA Cech & Altman-Discovered the catalytic properties of ribozymes, they are made of RNA Abbe- Optimized the microscope design (lens & condenser) Zeiss- Brought the lens resolution near limits of light Lacassagne- Developed autoradiography which uses light microscopy and imagers Coons-Developed fluorescent tagged antibodies st Ruska- Developed the 1 transmission electron microscope Palade & Porter- Developed the electron microscope stains for ultrastructure Robertson- Found the Unit membrane hypothesis Muhlethaler- Develops freeze fracture electron microscope G.Palade, 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 & 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 & Peroz- Alcohol precipitate of barley holds heat labile components - convert starch to sugars Kuhn- Coins term 'enzyme' “Greek "in leaven" Ducleaux- Uses suffix "ASE" for enzyme names Theories/Proven 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 th Reductionism- The primary methodological approach used in cell and molecular biology during the 20 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 Relativity-Albert Einstein- If speed of light is a constant, then time and motion are relative to the observer, and 2 mass and energy are equivalent E=MC Atomic Structure & Fission- Rutherford discovered atomic particles, Otto Hahn & 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 & 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 O2 Anaerobic- Type of Eubacteria, get energy from catabolism of foods without O2 Archaebacteria- original form oldest kind of cells, Methanogens- Type of Archarbacteria, converts CO2 + H2!CH4 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 µm 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 w/o 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 (CH2O)n 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) & 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 & deoxyribose) and a phosphate Pyrimidines- Cytosine (C), Thyamine (T), Uracil (U) Purines- Adenine (A), Guanine (G) Configuration- 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 (+,-) w/o 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) Forces- Electrostatic attraction based upon closeness of atoms involved in macromolecule interaction s for shape Hydrophobic/Hydrophilic Interactions- Repulsion of electrostatic dipoles of water by non-polars favors (fattyhydrocarbons) groups self-assembling substances that dissolve readily in water (ions & polars) water ? 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 ---> AcoA splitting of sugar 3) oxidation of AcoA ----> CO2 + NADH ------> H2O ADP + P ----> ATP Anabolism- Biosynthesis coupled reaction - energetically unflavored with favored Free Energy ∆G = ∆H - T∆S ∆G 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 Oxidation/Reduction- Redox Reactions e-/H+ are transferred between oxidized & reduced forms Oxidation - Removal of e- from a substrate Reduction - Gaining of e- (& often a proton as well, H+) Peptide bond- Formed by condensation reaction between amino of one amino acid & 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 & 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 & 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 & have marked N-Aaffinity for opiate receptors--compare endorphin Enzymes- Responsible for catalytic activity & function Transport Proteins- Bind & carry ligands Storage Proteins- Ex: ovalbumin, gluten & casein, ferretin Contractile (Motor)- Can contract, change shape, elements of cytoskeleton (actin, myosin, tubulin) Structural (support)- Collagen of tendons & cartilage, elastin of ligaments, keratin of hair, feathers, & nails, fibroin of silk & webs Defensive (protect)- Antibodies (IgG), fibrinogen & thrombin, snake venoms, bacterial toxins Regulatory (signal)- Regulate metabolic processes, hormones, transcription factors & enhancers, growth factor proteins Receptors (detect stimuli)- Light & 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 & 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 & connective tissue Collagen- Type of Scleroproteins has high Glycine, Proline, & no Cysteine, when boiled makes gelatin Keratins- Type of Scleroproteins, proteins of skin & 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, & transport proteins Glycoproteins- Type of complex proteins found in antibodies, cell surface proteins Nucleoproteins- Type of complex Proteins found in ribosomes & 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- Don't vary significantly in amino acids sequence examples: ubiquitin (mito) & 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 & chickens = 2 variants in yeast & horses = 48 variants Functional Groups -OH -NH2 -COOH -CH3 -C=O -SH -PO4 C-C(=O)-C C-C(=0)-NH2 -CH3 Hydroxyl Amine Carboxyl Methyl Carbonyl Sulfhydryl Phosphoryl Methyl = Alcohol = Amino Acid = Acid = Hydrocarbon = Aldehyde/Ketone = Disulfide = Phosphate =Ester =Amide =Hydrophobic Types of Reactions Functional group transfers Glu + ATP <---> G6P + ADP Redox reaction (oxidation/reduction) PGAld <---> 1,3di-PGA Rearrangement (isomerizations) G6P <---> Fruc6P C-C breaking or re-formation F1-6bP <---> DHAP + 3PGAld Condensations Protein(n) + aa ---> Protein(n+1) Hydrolysis Glu-Glu(n) + H2O ---> Glu-Glu(n-1) Classes of amino acids [classified... by R-Groups] Acidic - Negatively charged ASP & GLU R group with 2nd COOH that ionizes above pH 7.0 Basic - Positively charged LYS, ARG, HIS R group with 2nd amide that protonates below pH 7.0 Polar Uncharged SER, THR, TYR, ASN, GLN Are soluble in water, i.e., hydrophilic Non-Polar (aliphatic) GLY, ALA, VAL, LEU, ILE, PRO Contain only hydrocarbons R groups = hydrophobicity Aromatic (Hydrophobic non-polars) PHE,MET, TRP,CYS All contain R groups with ring structures Peptide Bond Characteristics Partial double bond character Shorter & stronger than C-C Longer, yet weaker than C=C No free rotation (group in same plane = TRANS), results is zig-zag planar molecule Protein shape & Conformation Native Protein Conformation- Is a 3-D spatial shape that's most thermodynamically stable, it has the lowest free energy expenditure & forms spontaneously Helix- A spiral staircase-like shape Fiber- Elongated bound monomers Globular- Roughly a sphere Physical forces-Non-covalent bonds, H-bonds, hydrophobic & hydrophilic interactions, & covalent bonds (as peptide bonds & disulfide bonds) Native Conformation of most enzyme proteins is globular-Interior is pocket of hydrophobic, exterior is hydrophilicmaximizes # H-bonds that form Chaperones- proteins that bind to others & 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 3.6 amino acid per turn Single repeat turn of helix (360 o ) = 0.54 nm Right handed helix - (counterclockwise) Helix formed from H-bond interactions H + of N (of any amino acid) & -O=C (of 4th amino acid) ¼ of amino acids in globular proteins occur in alpha helix Beta sheet- A linear extended zig-zag pleated sheet, formed by H-bonds intra- & inter-chain Domains- Structural units within larger peptides made of helix & sheets allowing maximal H-bonds Motifs- Combos of domains repeated in many different & 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 α helicies connected by 3 bends form a pocket for CoE & metal ion ligand binding sites ex: cytochrome B562 and TMV coat protein αβ Saddle- Sheets fold to make a core that looks like a saddle ex: LDH ββ Sandwich- Criss-cross patchwork of b b sheets that form a hydrophobic pocket ex: insecticyanin, antitrypsin ββ 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 3-D orientation of proteins in space Thermodynamically most stable conformation Weak non-covalent interactions & S-S bridges Hydrophobic interior & hydrophilic exterior Myoglobin MW 16,700 Cytochrome-C MW 12,400 - heme binding single polypeptide of 100 amino acids in ETS of mitochondria Lysozyme - MW 14,600 enzyme; egg white & human tears; 124 aa's with 4 S-S; that hydrolyses poly-saccharides in bacterial cell walls = bactericidal agent Ribonuclease - MW 13,700 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 Filaments- 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 & pestel, or tissue grinders, or blender, or sonicators with a buffered medium with substrate to rupture cells making homogenate Differential & Ultra-Centrifugation- Produces a pellet (solid) & 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 5-10%, while under a centrifuge, you will get sedimentation the smaller particle in the bottom and larger closer to the top Equilibrium Centrifugation- By bouyant density;density gradient centrifugation in 30% to 70% sucrose (or CsCl) cell part move up or down into bands, independent of size & 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 Affinity 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 (starch/polyacrylamide) separation is by size & 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, i.e., no charge 2-Dimensional Electrophoresis- Combines isoelectric focusing with SDS-electrophesis, if gel is turned at right angel & 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 fingerprint]- Treat a purified protein with proteolytic enzyme, Distinctive fragments electrophoresed or chromatographed Spectroscopy Spectroscopy- Measures intensity of light beam before & 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 aa's) Colorimetry - colored dye binds to amino acids Ninhydrin reaction - rx's 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 quantities...(10 -12 M) Quantification of protein present is based on BEER-LAMBERT Law linear relationship between light Absorbance vs. Concentration 1 (international) unit of Enzyme Activity- Amount of protein which converts 1 micromole of substrate to product per min at 25 0 C at optimal pH ex. Urease - 1 unit will liberate 1.0 mmole of ammonia from urea per minute at pH 7.0 at 25oC [equivalent to 1.0 I.U.] 1 unit Specific Activity- Units of enzyme activity per mg protein 1 unit Molecular Activity- Number of units of enzyme activity per µmole of enzyme Enzyme- Regulate metabolic reaction rates, control metabolism, molecules (mostly protein) that accelerate or catalyze chemical reactions (A--->B) 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 specific substrate,do not change direction (energetics) of reactions Reaction Path E + S <---> [ES] <---> 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 & 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 acid,etc 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 & 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 + -------> acetaldehyde + NADH Transferases- Catalyze the transfer of functional groups Hexokinase D-glu + ATP ----------> D-glu-6-P + ADP Hydrolyases- Catalyzes hydrolytic reactions adds water across C-C bonds Carboxypeptidase A [aa-aa]n + H 2 O ----> [aa-aa] n-1 + aa Lyases- Add or remove groups to C=C bonds Pyruvate decarboxylase pyruvate -----> acetaldehyde + CO 2 Isomerases [mutases]- Catalyze isomerizations Maleate isomerase maleate ----------> fumarate Ligases- Condensation of 2 substrates with splitting of ATP Pyruvate Carboxylase PYR + CO 2 + ATP ----> OAA + ADP + P Leonor Michaelis & Maude Menten Kinetics Proposed mathematical modeling of enzyme reactions, an algebraic expression of rectangular hyperbola k1 k3 E + S <--------->ES <--------> E + P k2 k4 Assumptions 1) Rate formation ES complex from E + P is negligible i.e., 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 there's more than 1 substrate, then each has its own Km measures "RELATIVE afffinity” of an enzyme for its substrate one enzyme with 2 substrates with following Km's - 0.1 M & 0.05 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 & M kinetics most regulatory enzymes (multi-subunits) are not treatable Enzyme Inhibition Irreversible- inhibitor molecule can not be easily removed from enzyme i.e, 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 & 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 & binds Extent of inhibition is concentration dependent, i.e., can be overcome if [S] is very high, i.e., [S] >>> [I] Classical example is malonic acid inhibition of SDH Easy to demonstrate is via Lineweaver-Burke plots Shows Vmax is SAME & 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, i.e., lowers total [E] Can NOT be overcome, even if [S] is very high Easy to demonstrate via Lineweaver-Burke plots Shows Km is SAME & 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 Form(of 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 ---> NADH + subst NADH ----> H+ proton motive force à ATP Photosynthetic Phosphorylation light + NADP ---> NADPH ----> H+ Cell Respiration Series cytoplasmic & mitochondrial, linked enzymatic pathways, stepwise oxidation of food molecules makes ATP Physiological view: uptake of O 2 & release of CO 2 Biochemical view: O2 consumption, CO2 production 3 Stages: 1. Digestion - food polymers --> monomers 2. Production of AcoA --> glycolysis & FAoxidation 3. Oxidation of AcoA to CO2 & H2 O --> KC & ETC Cellular Pathways: Glycolysis Glucose --> pyruvate + NADH + ATP Kreb's Cycle AcoA --> CO2 + NADH + GTP +FADH2 Electron Transport Chain (ETC) Passage of e's from NADH to O2 ---> H2 O + 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 --> 2 PYR + 4ATP (2 net) + 2NADH Energy investment phase (coupled Rx's) Phosphorylation of low energy intermediates Energy capture phase (fig 4.5p116) Redox reaction (glyceraldehyde3-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 e's 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 & Shuttles Krebs Cycle Krebs Cycle, Citric Acid Cycle, Tricarboxylic Acid Cycle- A cyclical biochemical pathway resulting in aerobic oxidation of cell fuels, as CH2 O, fatty acids, & amino acids, while making CO2 , H2 O, & ATP Overall reaction: Acetyl-CoA + 3NAD + E-FAD + GDP + P + 2H2 O --> CoASH + 3NADH + E-FADH2 + GTP + 2CO2 Enzymes of Krebs Cycle 5 Dehydrogenases- ISDH, a aKGDH, SDH, MDH, & PDH 2 Hydrolyases- aconitatse & fumarase 1 Thiokinase- succinyl thiokinase 1 Synthetase- citrate synthatase 2 multi-enzyme complexes each with 60 proteins & 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 coe's (3NADH & 1 FADH2 ) 2 CO2's are released 3 parts of Mitochondrial Oxidation of Pyruvate 1. PYR --> CO2 + H2 O --> NADH/FADH2 Krebs 2. e - of NADH/FADH2 --> O2 to make H2 O ETS 3. ADP + P ---> ATP Chemiosmosis Pyruvate Dehydrogenase Complex Oxidative decarboxylation of alpha-Keto acid Pyruvate Acetyl Co A HOOC-C-CH3 -----> CoA-S-C-CH3 + CO2 || || 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 1. CoASH Vitamin Pantothenate 2. Lipoate 3. Thiamine pyrophosphate 4. E-FAD + 5. NAD Lipoic acid Thiamin (B1) Riboflavin (B2) Niacin Regulation of Krebs Cycle Controls flow of intermediates [in & out] Substrate availability - mass action Allosteric inhibition - end product/feedback inhibition Covalent modification - reversible phosphorylation... Protein kinases & 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 <--> FAcoA + AMP + PP converts cyotplasmic FA to Fatty-acyl-coA 2. Carnitine acyl-transferase 1 (outer mito memb.) FattyCoA + carnitine <-> Fatty acyl-carnitine + CoASH transfers FAcoA to carnitine for transport across mitochondria 3. Carnitine acyl-tranferase 2 (in mitoplasm) Fatty acyl-carnitine + CoASH <--> 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 --> FADH2 b) hydration - addition of water c) dehydration w NAD --> 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 & Electron Transfer + Membrane = impermeant to most everything, esp to H outer membrane - porins - molecules 5,000 -10,000d inner membrane - 70% protein & 30% lipid... contains a. Redox proteins of ETC b. ATP synthase c. Carrier proteins- phosphate translocases, ADP/ATP translocases, pyruvate/H+ symporter d. Glycerol-P & malate shuttles Mitochondrial DNA and, 2 rRNA's + 16,500 np's, codes for 20% mito proteins subunits of NADH dehydrogenase, and 22 tRNA's, How Electron Transfer Works Redox Potential empirical measure of tendency to gain e's - strong reducing agent has negative - • Eo' - strong oxidizing agent has positive + • Eo’ ∆ Go' = -nf ∆ Eo’ NADH <---> NAD+ + H+ + 2e- -0.32V + H2O <---> ½ O2 + 2H + 2e +0.82V ∆Go' = -(1)(0.023) (1.14) = - 26.2 Kcal Electron Transfer Chain's Order --> 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 2e's and/or protons Flavoproteins FMN & FAD protein bound hydrogen carriers Iron sulfur proteins FeS non-heme iron electron carriers Ubiquinone CoQ - semiquinone & hydroquinone mobile membrane bound non-protein hydrogen carriers Cytochromes ( a, a3, b562, b566, c1, c) "colored proteins" 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 ATP Synthase hydrophilic channel for H+ flow + makes 100 ATP per 300 H per sec condenses ADP + Pi ---> ATP F0 – membrane piece & stalk F1 – soluble piece; 5 proteins Oxidative Phosphorylation- Making ATP Synthesis of ATP made via a proton motive gradient generated by transfer of e's to reduce O2 & make H2O through series of redox proteins Mechanism - Chemiosmotic Coupling Mitchell 1961 - a fundamental mechanism - arose early in evolution & was retained 3 steps ETC - passes e thru membrane carrier proteins PMF - Proton Motive Force gradient (pH difference) DpH 1.0 units ph 8.0 matrix vs. pH 7.0 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 & accessory pigments Capture electrons as reducing power in NADPH Reduction of CO 2 to CH 2O 2 Fundamental Reaction Mechanisms Light Reactions (photo-chemical 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 fixation (reduction) stages carboxylation CO2 RuBP --> 2 PGA reduction PGA + NADPH --> PGAL regeneration of RuBP via HMP path ---> RuBP 6CO 2 + 12H 2O* --> C 6H 12O 6 + 6H 2O + 6O* 2 Pigments Accessory Pigments Carotenoids- carotenes, xanthophylls Phycobilins- chromophore + a protein Phycoerythrin & phycocyanin Chlorophylls - a,b,c,d, 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 (e.g., O 2 evolution, APs) vs. the wavelength of light Molecular Excitation of Chlorophyll Absorption of Light Energy blue light [440nm] = 71.5 Kc/einstein red light [700nm] = 40.9 Kc/einstein Ground State- paired e's with opposite spin = stability absorption moves non-bounded e's to higher orbitals 1st 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 fluorescence emission of light of longer wavelength 700 nm --> 710nm in time frame 10-9sec less energetic 3. Re-radiated as phosphorescence emission of light much longer wavelength 700nm --> 720nm in real time (1sec) 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. non-cyclic) release of O2 capture of e- into coenzyme NADP + ---> NADPH ATPase makes ATP (just like in mitochondria) chemiosmosis & its location in chloroplasts Dark Reactions of Photosynthesis occur in stroma (chloroplasm) consume ATP and NADPH made in light reactions reduces (fixes) CO2 into CH2O (sugars) 3 different pathways to make sugar 1. C3 - CALVIN cycle 1 CO2 + 5C RuBP ---> (2) 3C sugars (PGA) (2) 3C sugars combine ---> 1 net glucose RuBP carboxylase [50% of leaf protein] Photo-respiration inhibition by O 2 2. C4 - Hatch & Slack pathway 1 CO 2 + 3C PGA --> 4C acid (mesophyll cells) 4C acid ---> 3C + CO 2 in 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 classified 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 fission-like process Leucoplasts- Defined via function Amyloplasts - synthsize & store starch Aleuoplasts - contain stored protein (crystals) Elaioplasts contain oil & fat globules - fat biosynthesis Chromoplasts- Found in flower petals, ripe fruit, scenescent leaves end point of plastids differentiation formed from leucoplasts & chloroplasts not proplastids contain numerous water soluble anthocyanin pigments of unknow function Differentiation - Elaboration of membrane, enzyme systems, & enlargement Proplastids --> chloroplasts, leucoplasts, chromoplasts Chloroplasts --> chloroplasts &/or chromoplasts Leucoplasts --> chloroplasts &/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 [400,000/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 DNA/chlp (equals bacterial cell DNA highly supercoiled & repetitive (6 copies) Enzymes of CO2 fixation and lipid droplets Thylakoid- Membranes ...
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