BSCI330 Exam 2 Review 03 08 2014 2 21 Lecture Enzymes Protein Evolution Greater evolutionary distance between two organisms fewer homologous proteins One amino acid change can alter proteins function a conservation of amino acids in protein domains with similar functions Rhodopsin visual pigments in insect and vertebrate eyes share 30 identical amino acids despite different structure Protein Families As genes evolve and duplicate proteins they code for diversify in structure and function But many proteins with a similar function have similar conserved motifs and form protein families Myosin super family Covalent Modifications to Proteins 1 Phosphorylation o addition of negatively charged phosphate group to R group of serine threonine tyrosine o Phosphate comes from ATP forming ADP o Catalyzed by protein kinases taken away by phosphatases o Phosphate group adds two negative charges that can participate in ionic bonds amino acid R groups or ions in solution Attractions strong enough to drive major structural changes solubility activity changes o Phosphate may create a new recognition site that allows other proteins to bind 2 Addition of sugars Glycosylation o Carbohydrate chains can be joined to OH group of serine O linked or NH2 group of aspargine N linked o Forms glycoproteins 3 Addition of lipids or glycolipids o addition of phospholipids and fatty acids to cysteine or an N terminal glycine residues to form lipoproteins o fatty acid chain can insert into hydrophobic core of biological membranes anchoring a protein to the membrane Enzymes Does not change G Required in small amounts Must be left unchanged at the end of a reaction so it can cycle back to bind more substrate Can increase rate of reaction by 10 8 to 10 12 fold Classification o Highly specific for their substrates can be categorized into families Oxy reductases Transferases transfer functional groups from one molecule to another Hydrolases catalyze hydrolysis of chemical bonds Lysases catalyze alteration or removal of a functional group Isomerases Ligases catalyze joining of two molecules together Kinases transfer phosphate groups Enzyme Substrate Binding o Binding occurs through reversible weak bonding to a stereo specific active site forms an enzyme substrate complex o Substrate then reacts to form an Enzyme Product complex then the product is released o Reaction of enzyme and substrate rapidly reaches steady state in which complex is stable and product is formed at a fixed rate Initial rate of product formation is determined by amount of Enzyme substrate complex formed Michaelis Menten kinetics o Km is the Michaelis constant and is approximately equal to the dissociation constant for the enzyme substrate complex o Vmax is the rate of product formation when the enzyme is saturated with substrate o Low Km enzyme has high affinity for subsrate o Rate Vmax S Km S o The value of Km lies within its substrates natural concentration range Mechanisms transition state reaction occurs activation energy o More than one substrate o Even after binding substrate the reaction still has to overcome an activation energy more catalysis is required through stabilization of reaction transition states o Almost ALL reactions proceed through a higher energy o As they react substrates will go through a transition state with a higher energy than either reactants or products before the o Energy needed to reach this transition state most of o Enzymes stabilize transition states lowering their energy and hence lowering the activation energy o Stabilizing Transition State Binding of substrate into an enzymes active site forces substrate into new conformation that more closely resembles that of the transition state This Induced Fit distorts and strains bonds with the substrate and lowers activation energy needed o Changing Substrate reactivity A Formation of temporary covalent bonds between enzyme R groups in active site and substrate will speed reaction B acceptance or donation of electrons and or protons between R groups cofactors and substrate functional groups may weaken some bonds and strengthen others thus speeding reaction C Ionic interaction repulsion or attraction of charged R groups with charged functional groups on substrate may weaken some bonds and speed reaction Cofactor ions can bind to substrate and cause it to temporarily acquire charge As a result product complex is formed Temporary covalent bonds are broken electrons protons are restored to enzyme or substrate Enzyme releases product o Prosthetic groups and cofactors Non protein molecules which aid catalysis In some cases these are covalently bound to enzyme Example organo metal compounds in which the metal can donate or accept electrons to from substrate Chromophores small organic molecules which absorb Cofactors and coenzymes metal ions Mg2 can function as aids in catalysis or binding of the substrate o Molecular tunnels and multi enzymes Some enzymes have more than one active site Product of one site forms the substrate for the next in a multi stage reaction In few cases substrates and products can sometimes tunnel through the protein as they are passed from one site to another creating a very efficient production light line Multi enzyme complexes allow efficient transfer or products between active sites although substance has to diffuse a short distance and does not tunnel 2 24 Lecture Methods of studying proteins and gene expression Enzyme Inhibitors Reversible Inhibitors common in nature o Competitive Reversibly bind to active site and compete with substrate Can be displaced by high S Increase Km lower affinity Do not reduce Vmax Non Competitive Reversibly bind away from active site to cause change in enzyme structure that lowers catalytic efficiency Lower Vmax Do not increase Km Do not affect substrate binding Allosteric inhibition o A form of non competitive inhibition o Seen in multi subunit enzymes made of catalytic subunits and a regulatory subunits o A small molecule the inhibitor binds to a site on the regulatory subunit o Results in change in regulatory subunits conformation which is coupled to a change in the catalytic subunits conformation reducing catalytic efficiency o For some allosteric enzymes small molecules activate rather than inhibit the enzymes Phosphorylation enzyme cascades o Phosphorylation of enzymes at specific sites by specific kinases can stimulate or inhibit their activity o Allows a kinase to regulate another o Chains cascades of regulated
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