Exam 2 Study Guide/Notes Mutations • Mutations involve the substitutions of amino acids o Conservative mutation: A polar R-group replaces another polar R-group o Non-conservative mutation: A polar or charged R-group replaces a non-polar or uncharged R-group • Mutations can occur randomly in nature and lead to evolution over long-term, OR can be engineered into proteins by engineering cells to express proteins with modified DNA sequences. Single-site mutation of a protein that dramatically alters its function: • Sickle cell disease is a common abnormality of the oxygen-binding protein, Hemoglobin • In this abnormality, a non-polar valine is Substituted for polar glutamate in Hemoglobin’s primary sequence to create the mutant HbS molecule. o This mutation exposes a hydrophobic patch on Hb’s surface which allows deoxygenated Hb molecules to polymerize and aggregate to form fibers of HbS molecules. o This distorts the red blood cell shape (greatly decreases surface area) and lowers it’s oxygen binding capacity Protein Engineering • “Site-directed mutagenesis”: A method used to make intentional and specific changes to the DNA sequence of a gene. o cDNA coding for a protein is increased by cloning it in bacterial cells and mutated within the cell (in vitro) o The mutated cDNA is expressed in a cell line or genetically-modified organism • Protein engineering is used to change a native protein’s characteristics to make it more useful in industry, research, or a medical purpose. Protein Evolution • The greater the evolutionary distance between two organisms, the fewer similar or identical amino acids in “homologous proteins” o Homologous proteins are the class of proteins that contain orthologs (same protein different species) and paralogs (proteins that arise from duplication) • Since even one amino acid change can alter function, there has been a conservation of amino acids in protein domains with similar functions throughout evolution. • Example: Rhodopsin (a chromophore)- the visual pigments in insect and vertabrate eyes have 30% identical amino acids in similar positions despite the difference in eye structure. (compound eye vs. single lens eye) Protein Families • As genes evolve and duplicate, the proteins they code for diversify in structure and function- even within a single organism. However, many proteins with a similar function have some similar conformed motifs, forming “protein families.” • Example: Myosin “super” family of motor proteins: Over 139 family members over many organisms that drive the movement of organelles, cytoskeleton, and musclesCovalent modifications to proteins 1. Phosphorylation (addition of phosphate) a. The addition of a negatively charged phosphate group to the R-group of serine, threonine, and tyrosine (All contain an –OH) i. Serine’s R-group is –CH2-OH ii. Serine Phosphorylated= Phosphoserine: -CH2-O-PO32- b. Phosphate usually comes from ATP, forming phosphorylated amino acid residue + ADP c. This reaction is catalyzed by a class of enzymes called protein kinases d. Many changes in protein structure and activity are driven by phosphorylation i. Each phosphate group adds two negative charges to the protein which can participate in new ionic bonds with close-by positively charged amino acid R-groups, or ions in solution. These attractions can be strong enough to drive major structural changes, activity changes, or changes in protein solubility. ii. The added phosphate group may create a new recognition site that allows other proteins to bind to the phosphorylated protein. e. (The addition and removal of a phosphate group can switch ON or OFF a certain protein) 2. Glycosylation (Addition of sugars) a. Carbohydrate chains (2-60 sugar monomers) can be joined to: i. OH groups of serine (linked to the oxygen) OR ii. NH2 groups of asparagine (linked to the Nitrogen) b. Glycoproteins are the products 3. Addition of lipids or glycolipids a. Addition of phospholipids and fatty acids to cysteine or an N-terminal glycine residues to form lipoproteins. b. The fatty acid chain anchors a protein to the membrane by inserting into the hydrophobic core of biological membranes. Enzymes • As a catalyst, an enzyme does not change ∆G for a reaction: They lower the activation energy required to start a reaction. o ∆G is the lowering in energy from the starting reactant to the finished product and is not affected by activation energy • They are required in small amounts • It is left unchanged at the end of the reaction in order to be reused and bind to more substrates. • They catalyze equally the forward and reverse reactions • Enzymes can increase reaction rates by 108-1012 fold. • Enzymes are highly specific for their substrates and classified into families depending on the class of reaction they catalyze: o Oxy-reductases (Redox Reactions) o Transferases- transfer functional groups from one molecule to another o Hydrolases- Catalyze hydrolysis of chemical bonds o Lyases- Catalyse alteration or removal of Functional Groupso Isomerases (amongst isomers) o Ligases- catalyze the joining of two molecules together o Kinases- transfer phosphate groups Enzyme reactions and substrate binding • Enzyme (E) functions begins when the substrate(s) (S) bind through reversible, weak bonding to a stereo-specific active site. • This creates an enzyme-substrate complex (ES) • The substrate in the complex reacts to form a product, which is released, yielding the free (and ready to function again) enzyme back. • When the E and S are mixed together in vitro, this reaction rapidly reaches a steady state in which the [ES] is stable and the product is produced at a fixed rate. o NOTE: Enzymes do not function at equilibrium, they function at a steady state • The initial rate of product formation is determined by the amount of ES formed. Rate = Vmax * [S]/(Km+[S]) • Vmax= rate of product formation when 100% of enzyme is present • A low Km = enzyme has high affinity for substrate • Km values are approximately equal to the dissociation constant Mechanisms • Binding multiple substrates together to the same surface speeds up a reaction immensely o However, even after binding, the reaction must overcome activation energy, so further catalysis is required through stabilization of transition states •
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