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 the DNA sequence of a gene within the cell in vitro Site directed mutagenesis A method used to make intentional and specific changes to o cDNA coding for a protein is increased by cloning it in bacterial cells and mutated 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 muscles Covalent 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 PO3 2 b Phosphate usually comes from ATP forming phosphorylated amino acid residue c This reaction is catalyzed by a class of enzymes called protein kinases ADP 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 Groups o 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 state 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 All reactions regardless of enzyme presence proceeds through a higher energy transition Transitional intermediate It is its own specific structure that forms between the reactants and the products of a reaction It is at a higher energy state than both reactants and products Enzymes stabilize transition states lowering their energy thus lowering activation energy for a reaction o Enzymes do not work at
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