Notes for Biochemistry

Biochemistry 1: Amino Acids, Peptides, and Protein * See appendix for full AA chart Amino Acids Found in Proteins Amino Acids: A molecule with 4 groups attached to a central ( a) carbon: an amino group, a carboxylic acid group, a hydrogen atom, and an R Group. The R Group determines function of that amino acid. Stereochemistry: The stereochemistry of the a -carbon is L for all chiral amino acids in eukaryotes. (carbohydrates are D-config). All chiral amino acids except cysteine have (S) configuration and all amino acids are chiral except for Glycine. Hydrophobic & Amino acids with long alkyl chains are hydrophobic. Hydrophilic: Those with charges are hydrophilic. All others fall in somewhere in between. and Protein Structure 1¡ Structure: Linear sequence of amino acids in a peptide. Stabilized by peptide bonds. The AA sequence is written N-terminus to Cterminus. N-terminus is POSITIVELY charged due to –NH3+. 2¡ Structure: The local structure of neighboring amino acid. Is stabilized by hydrogen bonding between amino groups and nonadjacent carboxyl groups. a -helices: A common 2¡ structure. Clockwise coils around a central axis. b-pleated A common 2¡ structure. Rippled strands that can be parallel sheets: or antiparallel. Proline: Can interrupt 2¡ structure because of its rigid cyclic structure. Structure of an Amino Acid Acid-Base Chemistry of Amino Acids Amphoteric: Amino acids can act as a base or an acid. pKa: The pH at which half of the species is deprotonated; [HA] = [A-]. pH: ¯pH Þ amino acid is fully protonated pH » pI Þ amino acid is a neutral zwitterion $pH Þ amino acid is fully deprotonated Isoelectric Point: (p9) The pH at which an amino acid is in zwitterion form; the charges cancel out to make a neutral molecule. !"#$ % &'()*+,-./(!. For no side chain: !: % . $ .;!" < !" = #$ #0 0 !"#0 % '1234./(! !"#5 % 6274.&8'23 $ For a NEUTRAL side chain: !: % . 0 .;!"#$ < !"#0= $ For a BASIC side chain: !: % . 0 .;!"#0 < !"#5= Note: Denaturing is when a protein (or nucleic acid) loses its 4¡, 3¡, and 2¡ structures due to breaking non-covalent interactions such as H-bonds, hydrophobic interactions, and dipole-dipole interactions. and Protein Structure 3¡ Structure: 3-D shape of a single polypeptide chain, and is stabilized by hydrophobic interactions, acid-base interactions, H-bonds, and disulfide bonds. Hydrophobic Push hydrophobic R groups to the interior or a Interactions: protein, which increases entropy of the surrounding water molecules and creates a negative Gibbs free energy. Disulfide Bonds: Occur when two cysteine molecules are oxidized and create a covalent bond between their thiol groups. This forms cystine. $ For an ACIDIC side chain: !: % . 0 .;!"#$ < !"#5= Titration: Midpoint: !> % !?# Equivalence Point: !> % !9 Peptide Bond Formation and Hydrolysis Terminology: Dipeptide: 2 residues Tripeptide: 3 residues Oligopeptides: Less than 20 residues Polypeptides: Greater than 20 residues Formation: Forming a peptide bond is a dehydration reaction. The nucleophilic amino group of one amino acid attacks the electrophilic carbonyl group of another amino acid. 4¡ Structure: The interaction between peptides in proteins that contain multiple subunits. Conjugated Proteins: Proteins with covalently attached molecules. Prosthetic Group: The attached molecule in a conjugated protein. Can be a metal ion, vitamin, lipid, carbohydrate, or nucleic acid. Denaturation: The loss of 3-D structure. Caused by heat or solute concentration. Polypeptide Structure Amide Bonds: The C-N bond of a peptide bond. Rigid due to resonance. Breaking: Breaking a peptide bond is a hydrolysis reaction. Peptide Bonds Biochemistry 2: Enzyme Enzymes as Biological Catalysts Effects of Local Conditions on Enzymes Enzymes: Biological catalysts that are unchanged by the reactions Temp and pH: they catalyze & are reusable. Enzymes DO NOT alter the DG or DH, nor the final equilibrium position. They only change the rate of reaction by altering the mechanism. Salinity: Catalyze both the FORWARD & REVERSE reactions. Exergonic Rxns: Release energy; DG is negative. Endergonic Rxns: Require energy; DG is positive. Oxidoreductases: REDOX reactions that involve the transfer of e-. Transferases: Move a functional group from one molecule to another. Hydrolases: Catalyze cleavage with the addition of H2O. Lyases: Catalyze cleavage without the addition of H 2O and without the transfer of e-. The reverse reaction (synthesis) is often more important biologically. Isomerases: Catalyze the interconversion of isomers, including both constitutional isomers and stereoisomers. Ligases: Join two large biomolecules, often of the same type. Lipases: Catalyze the hydrolysis of fats. Dietary fats are broken down into fatty acids and glycerol or other alcohols. Kinases: ADD a phosphate group. A type of transferase. Phosphatases: REMOVE a phosphate group. A type of transferase. Phosphorylases: Introduces a phosphate group into an organic molecule, notably glucose. Can affect an enzyme’s activity in vivo; changes in temperature and pH can result in denaturing of the enzyme and loss of activity do to loss of 2¡, 3¡, or 4¡ structure. In vitro, salinity can impact the action of enzymes. Regulation of Enzymes * See appendix for detailed information on enzyme inhibition Feedback Inhibition: An enzyme is inhibited by high levels of a product from later in the same pathway. Reversible The ability to replace the inhibitor with a compound Inhibition: of greater affinity or to remove it using mild laboratory treatment. Competitive When the inhibitor is similar to the substrate and Inhibition: binds at the active site, blocking the substrate from binding. Can be overcome by adding more substrate. Vmax is unchanged, Km increases. Uncompetitive When the inhibitor binds only with the enzymeInhibition: substrate complex. Vmax and Km both decrease. Noncompetitive When the inhibitor binds with equal affinity to the Inhibition: enzyme and the enzyme-substrate complex. Vmax decreases, Km is unchanged. Mixed Inhibition: When the inhibitor binds with unequal affinity to the enzyme and the enzyme-complex. Vmax decreases, Km is increased or decreased depending on if the inhibitor has a higher affinity for the enzyme or enzyme-substrate complex. Irreversible Alters the enzyme in such a way that the active site is Inhibition: unavailable for a prolonged duration or permanently. Enzyme Kinetics Saturation Kinetics: As ![S] Þ !rxn rate, until a max value is reached. Graphical Plots: Michaelis-Menten: Hyperbolic curve Lineweaver-Burk: Line Km The [S] at which an enzyme runs at half its Vmax. % ($% !" # $ &'$ ) %' Vmax: The maximum rate at which an enzyme can catalyze a reaction. This is when all enzyme active sites are saturated with substrate. Michaelis–Menten * # *",- $ ./0 + . /0 (%1 Equation: Cooperative Display a sigmoidal curve because of the change in Enzymes: activity with substrate binding. Mechanisms of Enzyme Activity Enzymes act by stabilizing the transition state, providing a favorable microenvironment, or bonding with the substrate molecules. Active Site: The site of catalysis. Suicide Inhibitor: A substrate analogue that binds IRREVSERIBLY to the active site via a covalent bond. Allosteric Effecter: Binds at the allosteric site and induces a change in the conformation of the enzyme so the substrate can no longer bind to the active site. Displays cooperativity, so it does not obey Michaelis-Menten kinetics. Positive Effectors: Exert a positive effect, !activity. Negative Effectors: Exert a negative effect, ¯activity. Homotropic Effector: An allosteric regulator that IS ALSO the substrate. Ex: O2 is a homotropic allosteric regulator of hemoglobin. Heterotropic Effector: An allosteric regulator molecule that is DIFFERENT from the substrate. Phosphorylation: Covalent modification with phosphate. Catabolism: Phosphorylated = active Anabolism: Phosphorylated = inactive Glycosylation: Covalent modification with carbohydrate. Zymogens: Precursor to an enzyme. Secreted in an inactive form and are activated by cleavage. Lock & Key Theory: The enzyme and substrate are exactly complementary and fit together like a key into a lock. Induced Fit Theory: The enzyme and substrate undergo conformational changes to interact fully. Cofactors: Metal cation that is required by some enzymes. Coenzyme: Organic molecule that is required by some enzymes. Reaction scheme for Michaelis-Menten enzyme activity. To simplify things, we assume that almost none of the product reverts back to ES, which is true at the start of the reaction. This is why K-2 is omitted from the reaction scheme shown below. Biochemistry 3: Nonenzymatic Protein Function and Protein Analysi Cellular Functions Protein Isolation Structural Proteins: Compose the cytoskeleton, anchoring proteins, and much of the extracellular matrix. The most common structural proteins are collagen, elastin, keratin, actin, and tubulin. They are generally fibrous in nature. Motor Proteins: Have one or more heads capable of force generation through a conformational change. They have catalytic activity, acting as ATPases to power mvmt. Common applications include muscle contraction, vesicle mvmt within cells, and cell motility. Examples include: myosin, kinesin, and dynein. Binding Proteins: Bind a specific substrate, either to sequester it in the body or hold its concentration at steady state. Cell Adhesion Allow cells to bind to other cells or surfaces. Molecules (CAM): Cadherins: Calcium dependent glycoproteins that hold similar cells together. Integrins: Have two membrane-spanning chains and permit cells to adhere to proteins in the extracellular matrix. Selectins: Allow cells to adhere to carbohydrates on the surfaces of other cells and are most commonly used in the immune system. * See appendix for detailed information Electrophoresis: Uses a gel matrix to observe the migration of proteins in responses to an electric field . Native PAGE: Maintains the protein’s shape, but results are difficult to compare because the mass / charge ratio differs for each protein. SDS-PAGE: Denatures the proteins and masks the native charge so that comparison of size is more accurate, but functional protein cannot be recaptured from the gel. Isoelectric Separates proteins by their isoelectric point (pI); the Focusing: protein migrates toward an electrode until it reaches a region of the gel where pH = pI of the protein. Chromatography: Separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase. Column Uses beads of a polar compound (stationary phase) with Chromatography: a nonpolar solvent (mobile phase). Ion-Exchange Uses a charged column and a variably saline eluent. Chromatography: Size-Exclusion Relies on porous beads. Larger molecules elute first Chromatography: because they are not trapped in the small pores. Affinity Uses a bound receptor or ligand and an eluent with free Chromatography: ligand or a receptor for the protein of interest. Antibodies: Immunoglobulins, Ig. Used by the immune system to target a specific antigen, which may be a protein on the surface of a pathogen or a toxin. The variable region is responsible for antigen binding. Biosignaling Protein Analysis Ion Channels: Can be used for regulating ion flow into or out of a cell. Ungated Channels: Always open. Voltage-Gated Channels: Open within a range of membrane potentials. Ligand-Gated Channels: Open in the presence of a specific binding substance, usually a hormone or neurotransmitter. Enzyme-Linked Participate in cell signaling through extracellular ligand Receptors: binding and initiation of 2nd messenger cascades. G Protein-Coupled GPCR has a membrane-bound protein called the GReceptors: Protein (a, b, g subunits). The 1st messenger ligand initiates the 2nd messenger and the cascade response. G-Protein Coupled Receptor Structure: Primarily determined through x-ray crystallography after the protein is isolated, although NMR can also be used. Amino Acid Determined using the Edman Degradation. Sequence: Concentration: Determined colorimetrically, either by UV spectroscopy or through a color change reaction. Bradford Assay, BCA Assay, and Lowry Reagent Assay each test for protein and have different advantages and disadvantages. The Bradford Protein Assay is most common. It uses a color change from brown-green ® blue. Beer-Lambert Law: !"#$%"&'() * + e+,+e+ * )./0'(/0$'+($)110(0)'/ - * 2&/3+4)'5/3+0'+(6 Notes: Epinephrine is a ligand 1st messenger. At the end of the GPCR process, Phosphodiesterase deactivates cAMP and GTP hydrolyzed back to GDP. , * ($'()'/%&/0$' Biochemistry 4: Carbohydrate Structure and Functio Carbohydrate Classification Nomenclature: 3 carbons: Trioses, 4 carbons: Tetroses, etc. Some common names: glucose, fructose & galactose. D and L: Based on the D- and L- forms of glyceraldehyde. Look at the highest numbered chiral carbon, -OH on right = Dsugars, -OH on left = L-sugars. Nearly all carbohydrates in nature are in the D-configuration. Compared to amino acids, which are found in the L-confiduration. Enantiomers: Stereoisomers that are non-superimposable mirror images of each other. D and L forms of the same sugar. Diastereomers: Any stereoisomer that is not an enantiomer. Epimers: Subtype of diastereomers that differ at exactly one chiral carbon. Anomers: A subtype of epimers that differ at the anomeric carbon. Monosaccharides Monosaccharides: Single carbohydrate units, with glucose as the most commonly observed monomer. Can undergo oxidation/reduction, esterification, and glycoside formation Aldoses: Oxidized into aldonic acids, reduced to alditols Sugar as Reducing Sugars that can be oxidized are reducing agents Agent: themselves. Can be detected by reacting with Tollen’s or Benedict’s reagents Deoxy Sugars: -H replaces –OH Esterification: Sugars react with carboxylic acids and their derivatives, forming esters Phosphorylation: A phosphate ester is formed by transferring a phosphate group from ATP onto the sugar. This rxn is similar to esterification Glycoside Formation: The basis for building complex carbohydrates and requires the anomeric carbon to link to another sugar Enantiomers Diastereomers Enantiomers Cyclic Sugar Molecules Complex Carbohydrates Cyclization: Describes the ring formation of carbohydrates from their straight-chain forms. Anomeric Carbon: The new chiral center formed in ring closure; it was the carbon containing the carbonyl in the straight-chain form. Disaccharides: Form as a result of glycosidic bonding between two monosaccharide subunits. Common examples: sucrose, lactose, maltose. Polysaccharides: Formed by repeated monosaccharide or polysaccharide glycosidic bonding. a -anomers: Have the –OH on the anomeric carbon trans to the free –CH2OH group. Cellulose: The main structural component for plant cell walls. Main source of fiber in human diet. b-anomers: Have the –OH on the anomeric carbon cis to the free – CH2OH group. Starches: Main energy storage form for plants. Amylose: Unbranched Amylopectin: Branched Haworth Represent 3D structure of a monosaccharide. Projections: Mutarotation: Spontaneous shift from one anomeric form to another with the straight-chain form as an intermediate. Maltose: (glucose + glucose with alpha glycosidic bond) Lactose: (galactose + glucose with beta glycosidic bond) b-D-Glucose a-D-Fructose Examples of Cyclic Sugar Molecules Sucrose: (glucose + fructose with alpha glycosidic bond) Biochemistry 5: Lipid Structure and Functio Structural Lipids Signaling Lipids Characteristics: Lipids are insoluble in water and soluble in nonpolar organic solvents. Phospholipids: Amphipathic and form the bilayer of membranes. Contain a hydrophilic (polar) head and hydrophobic (nonpolar) tails. The head is attached by a phosphodiester linkage, and determines the function of the phospholipid. Saturation: Saturation of the fatty acid tails determines the fluidity of the membrane. Saturated fatty acid = less fluid. Glycerophospholipids: Phospholipids that contain a glycerol backbone. Sphingolopids: Contain a sphingosine backbone. Many (but not all) sphingolipid are also phospholipids with a phosphodiester bond. These are sphingophospholipids. Sphingomyelins: The major class of sphingophospholipids and contain a phosphatidylcholine or phohsphatidylethanolamine head group. Part of the myelin sheath. Terpenes: Odiferous steroid precursors made from isoprene. One terpene unit (monoterpene) contains 2 isoprene units. Terpenoids: Derived from terpenes via oxygenation or backbone rearrangement. Odorous characteristics. Steroids: Contain 3 cyclohexane rings and 1 cyclopentane. Steroid Hormones: Have high-affinity receptors, work at low concentrations, and affect gene expression and metabolism. Cholesterol: A steroid important to membrane fluidity and stability; and serves as a precursor to many other molecules. Prostaglandins: Are autocrine and paracrine signaling molecules that regulate cAMP levels. Affect smooth muscle contraction, body temp, sleep-wake cycle, fever, pain. Vitamins A, D, E, & K: Fat soluble vitamins Vitamin A: Carotene, vision. Vitamin D: Cholecalciferol, bone formation. Vitamin E: Tocopherols, antioxidants. Vitamin K: Phylloquinone & menaquinones. Forms prothrombin, a clotting factor. Glycosphingolipids: Attached to sugar moieties instead of a phosphate group. Cerebrosides have 1 sugar connected to sphingosine. Globosides have 2 or more. Gangliosides: Contain oligosaccharides with at least 1 terminal Nacetylneuraminic acid (NANA). Waxes: Contain long-chain fatty acids esterified to longchain alcohols. Used as protection against evaporation and parasites in plants and animals. Steroid Energy Storage Phospholipid Polar head, nonpolar tails Triacylglycerols: Storage form of fatty acids. Contain 1 glycerol attached to 3 fatty acids by ester bonds. Very hydrophobic so do not carry additional water weight. Adipocytes: Animal cells used specifically for storage of large triacylglycerol deposits. Schematic of a phospholipid. May use glycerol or sphingosine for the backbone Free Fatty Acids: Unesterified fatty acids that travel in the bloodstream. Salts of free fatty acids are soaps. Saponification: The ester hydrolysis of triacylglycerols using a strong base like sodium or KOH. Micelle: Can dissolve a lipid-soluble molecule in its fatty acid core, and washes away with water because of its shell of carboxylate head groups. Cerebroside: A type of glycolipid. Any lipid linked to a sugar is a glycolipid Triacylglycerol Biochemistry 6: DNA and Biotechnolog Nucleic Acid Structure Nucleic acids are polymers of nucleotides. Types include Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA). 1¡ Structure: Linear sequence of nucleotides. 2¡ Structure: Interactions between bases within the same molecule. In DNA, the bases are held together by hydrogen bonds. 2¡ structure is responsible for the shape of nucleic acid. RNA 2¡ structure has 4 basic elements: Loops, helices, bulges, and junctions. Loops include stem-loops (hairpin loops), tetraloops, and psuedoknots. 3¡ Structure: The location of the atoms in 3D space. 4¡ Structure: Interactions of nucleic acids with other molecules. Example: Chromatin interacting with histones. DNA Replication * See appendix for full diagram DNA Replication: The process of producing an identical replica of a DNA molecule. Occurs in the S (synthesis) phase of the cell cycle. DNA Repair Oncogenes: Develop from mutations of proto-oncogenes, and promote cell cycling. May lead to cancer. Oncogenes = stepping on gas pedal Tumor Suppressor Code for proteins that reduce cell cycling or promote Genes: DNA repair. Mutated Tumor Suppressor genes = cutting the brakes Proofreading: DNA Polymerase proofreads its work and excises incorrectly matched bases. The daughter strand is identified by its lack of methylation and corrected accordingly. Mismatch Occurs during G2 phase using the genes MSH2 and MLH1. Repair: Nucleotide Fixes helix-deforming lesions of DNA such as Thymine Excision Repair: dimers. A cut-and-patch process. Excision Endonuclease. Nucleic Acid Structure: Includes DNA structure and RNA structure. Base Excision Fixes nondeforming lesions of the DNA helix such as Repair: cytosine deamination by removing the base, leaving apurinic/apyrimidinic (AP) site. AP Endonuclease then removes the damaged sequence, which can be filled in with the correct bases. Eukaryotic Chromosome Organization 46 chromosomes in human cells. DNA is wound around histone proteins to form nucleosomes, which may be stabilized by another histone protein. As a whole, DNA and its associated histones make up chromatin in the nucleus. Chromatin: Heterochromatin: Dark, dense, and silent Euchromatin: Light, uncondensed, and expressed DNA Structure * See appendix for full diagram DNA: Deoxyribonucleic Acid. A macromolecule that stores genetic information in all living organisms. Nucleoside: 5-carbon sugar + nitrogenous base. NO PHOSPHATE groups. Nucleotide: A nucleoside with 1 to 3 phosphate groups added. Nucleotides in DNA contain deoxyribose; in RNA they contain ribose. Adenine (A), Thymine (T), Guanine (G), Cytosine (C), Uracil (U). In RNA, U replaces T, so A pairs with U via 2 h-bonds. Watson-Crick Backbone of alternating sugar/phosphate groups. Always Model: read 5’ ® 3’. Two strands with antiparallel polarity wound into a double helix. Nitrogenous Purines: Adenine and Guanine. Made of two rings. Bases: Pyrimidines: Cytosine, Thymine, Uracil. Made of one ring. Chargaff’s Rules: # of Purines = # of Pyrimidines. A = T; C = G B-DNA vs Z-DNA: Most DNA is B-DNA, forming a right-handed helix. Low concentrations of Z-DNA, with a zigzag shape, may be seen with high GC-content or high salt concentration. Denature / Denatured: Pulled apart Reannealed: Reannealed: Brought back together Telomeres: Ends of chromosomes. Contain high GC-content to prevent unraveling of the DNA. During replication, telomeres are shortened, but this can be partially reversed by telomerase. Centromeres: Located in the middle of chromosomes and hold sister chromatids together until they are separated during anaphase in mitosis. High GC-content to maintain a strong bond between chromatids. Acrocentric When the centromere is located near one end of the Chromosome: chromosome and not in the middle. Recombinant DNA and Biotechnology Recombinant DNA: DNA composed of nucleotides from 2 different sources Hybridization: The joining of complementary base pair sequences. Polymerase Chain See appendix for details Reaction (PCR): Electrophoresis: See appendix for details Biochemistry 7: RNA and the Genetic Cod The Genetic Code Central Dogma: States that DNA is transcribed to RNA, which is translated to protein. Degenerate Code: Allows for multiple codons to encode for the same amino acid. Start / Stop Initiation (start): AUG Codons: Termination (stop): UAA, UGA, UAG Wobble: 3rd base in the codon. Allows for mutations to occur ? without effects in the protein. Wobble base pairings are less stable. Point Mutations: Silent: Mutations with no effect on protein synthesis. Usually found in the 3rd base of a codon. Nonsense (truncation): Mutations that produce a premature STOP codon. Missense: Mutations that produce a codon that codes for a DIFFERENT amino acid. Frameshift Result from a nucleotide addition or deletion, and Mutations: change the reading frame of subsequent codons. RNA: Similar to DNA except: Ribose sugar instead of deoxyribose. Uracil instead of thymine. Single stranded instead of double stranded. 3 Types of RNA: Messenger RNA (mRNA): Transcribed from DNA in the nucleus, it travels into the cytoplasm for translation. Transfer RNA (tRNA): Brings in amino acids and recognizes the codon on the mRNA using its anticodon. Ribosomal RNA (rRNA): Makes up the ribosome and is enzymatically active. Transcription Translation * See appendix for full diagram tRNA: Translates the codon into the correct amino acid. Ribosomes: Factories where translation (protein synthesis) occurs. Eukaryotes: 80s ribosomes Prokaryotes: 70s ribosomes Initiation: Prokaryotes: When the 30S ribosome attaches to the Shine-Delgarno Sequence and scans for a start codon; it lays down N-formylmethionine in the P site of the ribosome. Eukaryotes: When the 40S ribosome attaches to the 5’ cap and scans for a start codon; it lays down methionine in the P site of the ribosome. Elongation: The addition of a new aminoacyl-tRNA into the A site of the ribosome and transfer of the growing polypeptide chain form the tRNA in the P site to the tRNA in the A site. The now uncharged tRNA pauses in the E site before exiting the ribosome. The A site tRNA moves to the P site. Termination: Occurs when the codon in the A site is a stop codon; release factor places a water molecule on the polypeptide chain and thus releases the protein. Posttranslational Folding by chaperones. Formation of quaternary Modifications: structure. Cleavage of proteins or signal sequences. Covalent addition of other biomolecules (phosphorylation, carboxylation, glycosylation, prenylation). DNA Ligase: Fuse the DNA strands together to create one strand. * See appendix for full diagram Helicase: Unwinds the DNA double helix. Control of Gene Expression in Prokaryotes RNA Polymerase II: Binds to the TATA box within the promoter region of the gene (25 base pairs upstream from first transcribed base). hnRNA: Collective term for the unprocessed mRNA in nucleus. Posttranscriptional The process in eukaryotic cells where primary transcript Modification: RNA is converted into mature RNA. Introns cut out. Exons: Exit the nucleus and form mRNA. Introns: Spliced out so they stay in nucleus. Introns also enable alternative splicing. Alternative splicing: Usually introns are cut away and exons remain, but alternative splicing might change that. A certain exon may be cut out, or an intron may stay. This allows for the RNA segment to code for more than one gene. 5’ Cap and Poly-A tail are added to the mRNA. The cap and tail stabilize mRNA for translation. Prokaryotic cells can increase the variability of gene products from one transcript though polycistronic genes. There are multiple translation sites within the gene which leads to different gene products. Jacob-Monod Model: Explains how Operons work. Operons: Inducible or repressible clusters of genes transcribed as a single mRNA. Inducible Systems: Under normal conditions, they are bonded to a repressor. They are turned on when an inducer pulls the repressor off. Example: Lac operon. Repressible Systems: Transcribed under normal conditions; they can be turned off by a corepressor coupling with the repressor and the binding of this complex to the operator site. Example: Trp operon Control of Gene Expression in Eukaryotes Transcription Search for promoter and enhancer regions in the DNA, Factors: then bind to the DNA and recruit RNA polymerase. Promotors: Are within 25 base pairs of the transcription start site. Enhancers: Are more than 25 base pairs away from the transcription start site. Modification of chromatin structure affects the ability of transcriptional enzymes to access the DNA through histone acetylation (increases accessibility) or DNA methylation (decreases accessibility). Biochemistry 8: Biological Membrane Fluid Mosaic Model Accounts for the presence of lipids, protein, and carbohydrates in a dynamic, semisolid plasma membrane that surrounds cells Phospholipid Each phospholipid has a hydrophilic head and hydrophobic Bilayer: tail. They are arranged so the heads are facing outward and the tails make up the inside of the membrane. Proteins are embedded in the bilayer Lipid Rafts: Lipids move freely in the plane of the membrane and can assemble into lipid rafts Flippases: Specific membrane proteins that maintain the bidirectional transport of lipids between the layers of the phospholipid bilayer in cells Proteins & May also move within the membrane, but are slowed by Carbohydrates: their relatively large size Membrane Transport Concentration All transmembrane movement is based on Gradients: concentration gradients. The gradient tells us whether the process is passive or active Osmotic A colligative property. It is the pressure applied to a Pressure: pure solvent to prevent osmosis and is used to express the concentration of the solution. It can be conceptualized as a “sucking” pressure in which a solution is drawing water in, proportional to its concentration p! " #!$!%!& Passive Does not require energy because the molecule is moving Transport: down its concentration gradient. Simple Diffusion: A form of passive transport. Small, nonpolar molecules passively move form an area of high concentration to an area of low concentration until equilibrium is achieved Osmosis: A form of passive transport. Describes the diffusion of water across a selectively permeable membrane Facilitated A form of passive transport. Uses transport proteins to Diffusion: move impermeable solutes across the cell membrane Membrane Components Lipids: The primary membrane component, both by mass and mole fraction Triacylglycerols & Act as phospholipid precursors and are found in Fatty Acids: low levels in the membrane Glycerophospholipids: Replace one fatty acid with a phosphate group, which is often linked to other hydrophilic groups Cholesterol: Is present in large amounts and contributes to membrane fluidity and stability ¯temp = INCREASE fluidity #temp = DECREASE fluidity Waxes: Present in very small amounts, if at all; they are most prevalent in plants and function in waterproofing and defense Transmembrane A type of integral protein that spans the entire Proteins: membrane. They are often glycoproteins. Embedded Proteins: Are most likely part of a catalytic complex or involved in cellular communication Membrane-Associated May act as recognition molecules or enzymes Proteins: Carbohydrates: Can form a protective glycoprotein coat and also function in cell recognition Ligands: Extracellular ligands can bind to membrane receptors, which function as channels or as enzymes in second messenger pathways Gap Junctions: Allow for rapid exchange of ions and other small molecules between adjacent cells Tight Junctions: Prevent solutes from leaking into the space between cells via a paracellular route, but do not provide intercellular transport Desmosomes & Desmosomes bind adjacent cells by anchoring to Hemidesmosomes: their cytoskeletons. Hemidesmosomes are similar, but their main function is to attach epithelial cells to underlying structures Active Transport: Requires energy in the form of ATP or an existing favorable ion gradient Primary Active Uses ATP or another energy molecule to directly power Transport: the transport of molecules across a membrane Secondary Active “Coupled transport”. Harnesses the energy released by Transport: one particle going down its electrochemical gradient to drive a different particle up its gradient. Symport: Both particles flow the same direction Antiport: The particles flow in opposite directions Endocytosis & Methods of engulfing material into the cells or releasing Exocytosis: material out of the cell. Pinocytosis: Ingestion of liquids via vesicles Phagocytosis: Ingestion of larger solid materials Specialized Membranes Membrane (Vm) Maintained by the Na+/K+ pump and leak channels. Potential: Resting potential of most cells is between -40 and -80 mV Nernst The electrical potential created by one ion can be calculated Equation: using the Nernst Equation. ' "! (!) *!+ /0123 !,-!. /0123456789: < " ! =>?@ 8;789: * /0123 !,AB!. /0123456789:< 8;789: Goldman- Resting potential of a membrane at physiological temp can Hodgkin-Katz be calculated using the Goldman-Hodgkin-Katz Voltage Voltage Eq: Equation, which is derived from the Nernst equation. O!H K L!/QK 3456789:OHRST !L!/UVT38;789: ! L!/MNK3 H 456789: P CD " EF?G!,AB!. IJK H K L!/MNK 3 O!H IJ 8;789: PK L!/Q K3 8;789: OHRST !L!/UV T3 < 456789: Mitochondrial The outer mitochondrial membrane is highly permeable to Membranes: metabolic molecules and small proteins. The inner mitochondrial membrane surrounds the mitochondrial matrix, where the citric acid cycle produces electrons used in the ETC. The inner mito membrane does not contain cholesterol. Biochemistry 9: Carbohydrate Metabolism I – Glycolysis, Glycogen, Gluconeogenesis, and the Pentose Pathwa Glucose Transport GLUT-2: Found in liver (for glucose storage) and pancreatic b-islet cells (as part of the glucose sensor). Has "Km GLUT-4: Found in adipose tissue and muscle. Stimulated by insulin. Has ¯Km Glycogenesis and Glycogenolysis * See appendix for full diagram Glycogenesis: The production of glycogen using two main enzymes: Glycogen Synthase, and Branching Enzyme. Occurs in the liver and muscle cells. Glycogen is stored in liver. Glycogen Synthase: Creates a-1,4 glycosidic bonds between glucose. Branching Enzyme: Creates branches with a-1,6 glycosidic bonds. Glycogenolysis: The breakdown of glycogen using two main enzymes: Glycogen Phosphorylase, and Debranching Enzyme. Glycolysis * See appendix for full diagram Glucose + 2NAD+ + 2ADP + 2P ® 2Pyruvate + 2ATP + 2NADH + 2H+ Important enzymes: Glucokinase: Converts glucose to glucose 6-phosphate in the (irreversible) pancreatic b-islet cells as part of the glucose sensor. Hexokinase: Converts glucose to glucose 6-phosphate in in (irreversible) peripheral tissues. Inhibited by its product G 6-P. Phosphofructokinase-1: PFK-1. Phosphorylates fructose 6-phospate to (irreversible) fructose 1,6-bisphosphate in the rate-limiting step. Activated by AMP and fructose 2,6bisphosphate. Inhibited by ATP and citrate. Phosphofructokinase-2: PFK-2. Produces fructose 2,6-bisphosphate that activates PFK-1. It is activated by insulin; inhibited by glucagon. Glyceraldehyde-3-phosphate dehydrogenase: Produces NADH, which can feed into the electron transport chain. Glycogen Removes single glucose 1-phosphate molecules by Phosphorylase: breaking a-1,4 glycosidic bonds. In the liver, it is activated by glucagon to prevent low blood sugar. In exercising skeletal muscle, it is activated by epinephrine and AMP to provide glucose for the muscle itself. Debranching Moves a block of oligoglucose from the branch and Enzyme: connects it to the chain using an a-1,4 glycosidic bond. It also removes the branchpoint, which is connected via an a-1,6 glycosidic bond, releasing a free glucose molecule. Gluconeogenesis * See appendix for full diagram Occurs in both the cytoplasm and mitochondria, predominantly in the liver with a small contribution from the kidneys. Most gluconeogenesis is simply the reverse of glycolysis, using the same enzymes. The 3 irreversible steps of glycolysis must be bypassed by different enzymes. Pyruvate Kinase: Perform substrate-level phosphorylation, placing (irreversible) an inorganic phosphate onto ADP to form ATP. Pyruvate Converts pyruvate to oxaloacetate, which is converted to Carboxylase: PEP by PEPCK. Together, these two enzymes bypass pyruvate kinase. Pyruvate carboxylase is activated by Acetyl-CoA. PEPCK is activated by glucagon and cortisol. The NADH produced in glycolysis is oxidized by the mitochondrial electron transport chain when O2 is present. If O2 or mitochondria are absent, the NADH produced in glycolysis is oxidized by cytoplasmic lactate dehydrogenase. Examples include RBCs and skeletal muscle. Fructose-1,6- Converts fructose 1,6-bisphosphate to fructose 6bisphosphatase: phosphate, bypassing phosphofructokinase-1. This is the rate-limiting step of gluconeogenesis. It is activated by ATP and glucagon. Inhibited by AMP and insulin. Pyruvate Dehydrogenase A complex of enzymes that convert pyruvate to Acetyl-CoA right before the citric acid cycle. It is stimulated by insulin and inhibited by acetyl-CoA. The Pentose Phosphate Pathway Also known as the hexose monophosphate (HMP) shunt, it occurs in the cytoplasm of most cells. Glucose 6-Phosphate enters the pathway and the products are NADPH, sugars for biosynthesis, and glycolysis intermediates. Rate-Limiting Glucose-6-phosphate dehydrogenase (G6PD), which is Enzyme: activated by NADP+ and insulin and inhibited by NADPH. Other Monosaccharides Galactose: Comes from lactose in milk. Trapped in the cell by galactokinase, and converted to 1-phosphate via galactose-1phosphate uridyltransferase and an epimerase. Fructose: Comes from honey, fruit, and sucrose. Trapped in the cell by fructokinase, then cleaved by aldolase B to form glyceraldehyde and DHAP. Biochemistry 10: Carbohydrate Metabolism I – Aerobic Respiratio Acetyl-CoA Acetyl-CoA: Contains a high-energy thioester bond that can be used to drive other reactions when hydrolysis occurs. Acetyl-CoA Formation: Can be formed from fatty acids, which enter the mitochondria using carriers. The fatty acid couples with CoA in the cytosol to form fatty acyl-CoA, which moves to the intermembrane space. The acyl (fatty acid) group is transferred to carnitine to form acyl-carnitine, which crosses the inner membrane. The acyl group is transferred to a mitochondrial CoA to re-form fatty acylCoA, which can undergo b-oxidation to form acetyl-CoA. Can also be formed from the carbon skeletons of ketogenic amino acids, ketone bodies, and alcohol. Pyruvate Dehydrogenase Oxidizes pyruvate, creating CO2; it requires thiamine (PDH): pyrophosphate (vitamin B1, TPP) and Mg 2+. Dihydrolipoyl Oxidizes the remaining two-carbon molecule using lipoic Transacetylase: acid, and transfers the resulting acetyl group to CoA, forming acetyl-CoA. Dihydrolipoyl Uses FAD to reoxidize lipoic acid, forming FADH 2. This Dehydrogenase: FADH2 can later transfer electrons to NAD+, forming NADH that can feed into the electron transport chain. Pyruvate Dehydrogenase Phosphorylates PDH when ATP or acetyl-CoA levels are Kinase: high, turning it off. Pyruvate Dehydrogenase Dephosphorylates PDH when ADP levels are high, turning Phosphatase: it on. Reactions of the Citric Acid Cycle * See appendix for full diagram Citric Acid Takes place in the mitochondrial matrix. Its main purpose is to Cycle: oxidize carbons in intermediates to CO 2 and generate high-energy electron carriers (NADH and FADH2) and GTP. Citrate Couples acetyl-CoA to oxaloacetate and then hydrolyzes the Synthase: resulting product, forming citrate and CoA-SH. This enzyme is regulated by negative feedback from ATP, NADH, succinyl-CoA and citrate. Aconitase: Isomerizes citrate to isocitrate. Isocitrate Oxidizes and decarboxylates isocitrate to form a-ketoglutarate. Dehydrogenase: This enzyme generates the first CO2 and first NADH of the cycle. As the rate-limiting step of the citric acid cycle, it is heavily regulated: ATP and NADH are inhibitors; ADP and NAD+ are activators. a -Ketoglutarate Acts similarly to PDH complex, metabolizing a-ketoglutarate to Dehydrogenase form succinyl-CoA. This enzyme generates the second CO2 and Complex: second NADH of the cycle. It is inhibited by ATP, NADH, and succinyl-CoA; it is activated by ADP and Ca2+. Succinyl-CoA Hydrolyzes the thioester bond in succinyl-CoA to form succinate Synthesis: and CoA-SH. This enzyme generates the one GTP generated in the cycle. Succinate Oxidizes succinate to form fumarate. This flavoprotein is anchored Dehydrogenase: to the inner mitochondrial membrane because it requires FAD, which is reduced to form the one FADH 2 generated in the cycle. Fumarase: Hydrolyzes the alkene bond of fumarate, forming malate. Malate Oxidizes malate to oxaloacetate. This enzyme generates the third Dehydrogenase: and final NADH of the cycle. The Electron Transport Chain Acyl Carnitine Translocase: Mechanism for Acyl CoA to enter the mitochondrial matrix. The mitochondrial matrix is where Acyl CoA can undergo b-oxidation to form Acetyl-CoA. * See appendix for full diagram Electron Transport Takes place on the matrix-facing surface of the inner Chain: mitochondrial membrane. NADH donates electrons to the chain, which are passed from one complex to the next. As the ETC progresses, reduction potentials increase until oxygen, which has the highest reduction potential, receives the electrons. Complex I: NADH-CoQ Oxidoreductase. Uses an iron-sulfur cluster to transfer electrons from NADH to flavin mononucleotide (FMN), and then to CoQ, forming CoQH2. 4 H+ ions are translocated by Complex I. Complex II: Succinate-CoQ Oxidoreductase. Uses an iron-sulfur cluster to transfer electrons from succinate to FAD, and then to CoQ, forming CoQH2. No H+ pumping occurs at complex II. Oxidative Phosphorylation Proton-Motive The electrochemical gradient generated by the electron transport Force: chain across the inner mitochondrial membrane. The intermembrane space has a higher concentration of protons than the matrix; this gradient stores energy, which can be used to form ATP via chemiosmotic coupling . ATP Synthase: The enzyme responsible for generating ATP from ADP and Pi F0 Portion: An ion channel, allowing H+ to flow down the gradient from the intermembrane space to the matrix F1 Portion: Uses the energy released by the gradient to phosphorylate ADP into ATP. Complex III: CoQH2-Cytochrome C Oxidoreductase. Uses an iron-sulfur cluster to transfer elcetrons form CoQH 2 to heme, forming cytochrome C as part of the Q cycle. 4 H+ ions are translocated by complex III. Complex IV: Cytochrome C Oxidase. Uses cytochromes and Cu 2+ to transfer electrons in the form of hydride ions (H -) from cytochrome c to oxygen, forming water. 2 H+ ions are translocated by complex IV. NADH cannot cross the inner mitochondrial membrane. Therefore, one of two available shuttle mechanisms to transfer electrons in the mitochondrial matrix must be used. Glycerol 3-Phosphate Shuttle: Malate-Aspartate: Electrons are transferred from NADH to DHAP, forming glycerol 3-phosphate. These electrons can then be transferred to mitochondrial FAD, forming FADH 2. Electrons are transferred from NADH to oxaloacetate, forming malate. Malate can then cross the inner mitochondrial membrane and transfer the electron to mitochondrial NAD+, forming NADH. Biochemistry 11: Lipid and Amino Acid Metabolism Lipid Digestion and Absorption Mechanical Mechanical digestion of lipids occurs primarily in the Digestion: mouth and stomach. Chemical Chemical digestion of lipids occurs in the small intestine Digestion: and is facilitated by bile, pancreatic lipase, colipase, and cholesterol esterase. Emulsification: Upon entry into the duodenum, emulsification occurs, which is the mixing of two normally immiscible liquids; in this case, fat and water. (A common example of an emulsion is oil-and-vinegar salad dressing). This increases the surface area of the lipid, which permits greater enzymatic interaction and processing. Emulsification is aided by bile salts. Micelles: Water-soluble spheres with a lipid-soluble interior. Digested lipids may form micelles to be carried to the intestinal epithelium where they are absorbed across the plasma membrane. Short vs. Long Short-chain fatty acids are absorbed across the intestine Chain Fatty Acids: into the blood. Long-chain fatty acids are absorbed as micelles and assembled into chylomicrons for release into the lymphatic system. Fatty Acids and Triacylglycerols Fatty Acids: Carboxylic acids with a long chain Saturated: No double bonds Unsaturated: One or more double bonds Fatty Acid Synthesized in cytoplasm from acetyl-CoA transported out of Synthesis: the mitochondria. Five steps: Activation, bond formation, reduction, dehydration, and a second reduction. Arachidonate: Precursor to eicosanoid signaling molecules: prostaglandins, prostacyclins, & thromboxanes. Also precursor to leukotrienes. Fatty Acid Oxidation occurs in the mitochondria following transport by the Oxidation: carnitine shuttle. b-oxidation uses cycles of oxidation, hydration, oxidation, and thiolysis cleavage. The fatty acid chain is shortened by two carbon atoms. FADH2, NADH, and acetyl CoA are generated. The carboxylic acid is the a end. The w carbon counting starts on the other end. Cis-Oleate, a cis-D 9 fatty acid The cis bond prevents tight packing, which lowers the melting point. a -Linolenate, an Omega-3 Fatty Acid (3rd carbon from the w end) Ketone Bodies Ketogenesis: Ketone bodies form via ketogenesis due to excess acetylCoA in the liver during a prolonged starvation state Ketolysis: Regenerates acetyl-CoA for use as an energy source in peripheral tissues Lipid Mobilization and Transport Lipid Lipids are mobilized from adipocytes by hormone-sensitive Mobilization: lipase. Lipids are mobilized from lipoproteins by lipoprotein lipase. Chylomicrons: Transport dietary triacylglycerols, cholesterol, & cholesteryl esters from intestine to tissues. Uses the lymphatic system. Lipoproteins: The transport mechanism for lipids. Very-low-density: Liver ® tissues. Intermediate-density: Transition particle. VLDL ® IDL ® VLDL Low-density: Bad. Moves cholesterol ® tissues. High-density: Good. Moves cholesterol ® liver, exits body. Apolipoproteins: Form the protein component of lipoproteins. They are receptor molecules that control interactions between li t i Energy Source: The brain can derive up to 2/3 of its energy from ketone bodies during prolonged starvation Protein Catabolism Protein digestion occurs primarily in the small intestine. Catabolism of cellular proteins occurs only under conditions of starvation. Amino acids released from proteins usually lose their amino group through deamination. The remaining carbon skeleton can be used for energy. Glucogenic Can be converted into glucose through gluconeogenesis. Amino Acids: All but leucine and lysine. Ketogenic Can be converted into acetyl-CoA and ketone bodies. Amino Acids: Leucine and lysine are the only amino acids that are solely ketogenic. Cholesterol Metabolism Cholesterol may be obtained through dietary sources or through de novo synthesis in the liver HMG-CoA Synthesizes mevalonate. This is the rate limiting step of Reductase: cholesterol synthesis LCAT: Catalyzes the formation of cholesteryl esters for transport with HDL CETP: Catalyzes the transition of IDL to LDL by transferring cholesteryl esters from HDL Fates of the amino acid carbon skeleton following protein catabolism Biochemistry 12: Bioenergetics and Regulation of Metabolism Thermodynamics and Bioenergetics Hormonal Regulation of Metabolism Open System: Matter & energy can be exchanged with the environment Closed System: Only energy can be exchanged with the environment. No work is performed because pressure and volume remain constant. -./012345 $ ' -6/0.7/23'./.785 $ 1.20'.9:12/8. Entropy: A measure of energy dispersion in a system Insulin: Secreted by pancreatic b-cells, regulated by glucose ¯blood glucose by increasing cellular uptake "rate of anabolic metabolism Glucagon: Secreted by pancreatic a -cells, stimulated by low glucose and high amino acid levels "blood glucose by promoting gluconeogenesis and glycogenolysis in the liver Change in Free Standard Free Energy, DG¡: The energy change that occurs Energy: at 1 M concentration, 1 atm, and 25¡C. Modified Standard State, DG¡’: Indicates physiological conditions. ;<=> $ ?&@AB, so pH is 7. Glucocorticoids: "blood glucose in response to stress by mobilizing fat stores and inhibiting glucose uptake. They increase the impact of glucagon and catecholamines. Ex: cortisol The Role of ATP Catecholamines: Promote glycogenolysis and "basal metabolic rate through sympathetic nervous system activity. “Adrenaline rush”. Ex: epinephrine and norepinephrine ATP structure ATP is a mid-level energy molecule. It contains high-energy phosphate bonds that are stabilized upon hydrolysis by resonance, ionization, and loss of charge repulsion. Energy Source: ATP provides energy through hydrolysis and coupling to () energetically unfavorable reactions. !"# $ %&' Thyroid "basal metabolic rate, as evidenced by "O2 consumption Hormones: and heat production when they are secreted. T3 is more potent than T4, but has a shorter half-life and is available in lower concentrations in the blood. T4 is converted to T3 at the tissues. Thyroid hormones are tyrosine-based. *+, Phosphoryl ATP can donate a phosphate group to other molecules. Group Transfers: For example, in Glycolysis, it donates a Phosphate group to glucose to form glucose 6-phosphate Biological Oxidation and Reduction Biological oxidation and reduction reactions can be broken down into component half-reactions. Half-reactions provide useful information about stoichiometry and thermodynamics High Energy May be soluble or membrane-bound. Includes NADH, Electron Carriers: NADPH, FADH2, ubiquinone, cytochromes, and glutathione. Flavoproteins: A subclass of electron carriers that are derived from riboflavin (vitamin B2). Examples: FAD and FMN Metabolic States Equilibrium: Equilibrium is an undesirable state for most biochemical reactions because organisms need to harness free energy to survive. Postprandial State: Well-fed, absorptive. "Insulin. Anabolism prevails. Postabsorptive State: Fasting. ¯Insulin. "glucagon and catecholamine. Transition to catabolism. Prolonged Fasting: Starvation. ""glucagon and catecholamine. Most tissues rely on fatty acids. 2/3 of brain activity can be derived from ketone bodies. Integrative Metabolism Tissue-Specific Metabolism Liver: The most metabolically diverse tissue. Hepatocytes are responsible for the maintenance of blood glucose levels by glycogenolysis and gluconeogenesis in response to pancreatic hormone stimulation. The liver also processes lipids and cholesterol, bile, urea, and toxins. Adipose Tissue: Stores lipids under the influence of insulin and releases them under the influence of epinephrine. Skeletal Muscle: Skeletal muscle metabolism will differ depending on current activity level and fiber type. Resting muscle: Conserves carbohydrates in glycogen stores and uses free fatty acids from the bloodstream. Calorimetry: Measures metabolic rates Respiratory RQ. Estimates the composition of fuel that is actively Quotient: consumed by the body. CD $ ' EFG'HI+JKLMJ F 'L+NOK*MJ G Regulatory Ghrelin: "appetite. (sight, sound, taste, smell of food) Hormones: Orexin: "appetite. Leptin: ¯appetite by suppressing orexin production Body Mass Index: PQR $ ' *SOO TMUVTWG Active muscle: May use anaerobic metabolism, oxidative phosphorylation of glucose, direct phosphorylation from creatine phosphate, or fatty acid oxidation, depending on fiber type and exercise duration. Cardiac Muscle: Uses fatty acid oxidation in both the well-fed and fasting states. Brain and Consume only glucose in all metabolic states, except for Nervous Tissue: prolonged fasts, where up to 2/3 of the brain’s fuel may come from ketone bodies.