2/21 Lecture-EnzymesProtein EvolutionGreater evolutionary distance between two organisms fewer homologous proteinsOne amino acid change can alter proteins function- a conservation of amino acids in protein domains with similar functionsRhodopsin- visual pigments in insect and vertebrate eyes- share 30% identical amino acids despite different structureProtein FamiliesAs 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” familyCovalent Modifications to Proteins1)Phosphorylationaddition of negatively charged phosphate group to R-group of serine, threonine, tyrosinePhosphate comes from ATP, forming ADP+Catalyzed by protein kinases (taken away by phosphatases)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 changesPhosphate may create a new recognition site that allows other proteins to bind2) Addition of sugars- GlycosylationCarbohydrate chains can be joined to OH group of serine (O-linked) or NH2 group of aspargine (N-linked)Forms glycoproteins3) Addition of lipids or glycolipidsaddition of phospholipids and fatty acids to cysteine or an N-terminal glycine residues to form lipoproteinsfatty acid chain can insert into hydrophobic core of biological membranes, anchoring a protein to the membraneEnzymesDoes not change ΔGRequired in small amountsMust be left unchanged at the end of a reaction so it can cycle back to bind more substrateCan increase rate of reaction by 10^8 to 10^12 foldClassificationHighly specific for their substrates, can be categorized into familiesOxy- reductasesTransferases- transfer functional groups from one molecule to anotherHydrolases- catalyze hydrolysis of chemical bondsLysases- catalyze alteration or removal of a functional groupIsomerasesLigases- catalyze joining of two molecules togetherKinases- transfer phosphate groupsEnzyme-Substrate BindingBinding occurs through reversible, weak bonding to a stereo-specific active site forms an enzyme substrate complexSubstrate then reacts to form an Enzyme Product complex, then the product is releasedReaction 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 formedMichaelis-Menten kineticsKm is the Michaelis constant and is approximately equal to the dissociation constant for the enzyme substrate complexVmax is the rate of product formation when the enzyme is saturated with substrateLow Km- enzyme has high affinity for subsrateRate = (Vmax*[S])/(Km + [S])The value of Km lies within its substrates natural concentration rangeMechanismsMore than one substrateEven after binding substrate the reaction still has to overcome an activation energy- more catalysis is required through stabilization of reaction transition statesAlmost ALL reactions proceed through a higher energy transition stateAs they react substrates will go through a transition state with a higher energy than either reactants or products before the reaction occursEnergy needed to reach this transition state most of activation energyEnzymes stabilize transition states, lowering their energy and hence lowering the activation energyStabilizing Transition StateBinding 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 neededChanging Substrate reactivityA) Formation of temporary covalent bonds between enzyme R-groups in active site and substrate will speed reactionB) 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 reactionC) 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 chargeAs a result, product complex is formed. Temporary covalent bonds are broken, electrons/protons are restored to enzyme or substrateEnzyme releases productProsthetic groups and cofactorsNon protein molecules which aid catalysis. In some cases these are covalently bound to enzymeExample- organo-metal compounds in which the metal can donate or accept electrons to/from substrateChromophores- small organic molecules which absorb lightCofactors and coenzymes- metal ions (Mg2+) can function as aids in catalysis or binding of the substrateMolecular tunnels and multi-enzymesSome 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 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 expressionEnzyme InhibitorsReversible Inhibitors- common in natureCompetitiveReversibly bind to active site and compete with substrate. Can be displaced by high [S]Increase Km- lower affinityDo not reduce VmaxNon-CompetitiveReversibly bind away from active site to cause change in enzyme structure that lowers catalytic efficiencyLower VmaxDo not increase Km. Do not affect substrate bindingAllosteric inhibitionA form of non-competitive inhibitionSeen in multi-subunit enzymes, made of “catalytic subunits” and a “regulatory subunits”A small molecule (the inhibitor) binds to a site on the regulatory subunitResults in change in regulatory subunits conformation which is coupled to a change in the catalytic subunits conformation, reducing catalytic efficiencyFor some allosteric enzymes, small molecules activate, rather than inhibit the enzymesPhosphorylation- enzyme cascadesPhosphorylation of enzymes at specific sites, by specific kinases can stimulate or inhibit their activityAllows a kinase to regulate
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