Lectures 6-8 BCM 475 - M001
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BCM 475 - M001
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Lecture 914 Experimental Techniques 1 p 79104 Ultracentrifugation Technique used to purify proteins s m 1 v p f s sedimentation coefficient m mass of the particle V bar partial specific volume p density of the medium f frictional coefficient measure of the shape of the particle The Importance of an Amino Acid Sequence An amino acid sequence can serve as a starting point for determining the structure and function of a novel protein by comparing the known amino acid sequence with all other known sequences Amino acid sequences of the same protein present in different species can be compared to determine each species distinct evolutionary pathways Amino acid sequences can be used to discover internal repeats within a protein Specific amino acid sequences in many proteins serve as signals designating their destinations or controlling their processingquot Amino acid sequences can be used to function as an antigen in an organism and subsequently lead to the production of antibodies Amino acid sequences can be used to construct DNA sequences that may subsequently used as a DNA probe to further determine the full sequence of the protein of interest Steps For Determining Amino Acid Sequences of Proteins by Automated Edman Degradation 1 Determine the amino acid composition Heat peptide to hydrolyze into amino acids Separate obtained constituent amino acids by ionexchange chromatography and determine amino acid composition by analyzing elution profiles for the amino acids The amount of each amino acid present is determined from the absorbancequot eg on sulfonated polystyrene resin acidic amino acids will ow out of column first React with ninhydrin or uorescamine indicator dyes Amino acid will conjugate to the indicator dye and the intensity of the color will indicate the different concentrations of each amino acid 2 Identify the Nterminal amino acid React amino acid with phenyl isothiocyanate Edman degradation to form a phenylthiocarbamoyl derivative with a labeled aminoterminal residue Place phenylthiocarbamoyl derivative in a mildly acidic solution A cyclic derivative phenylthiohydantoin PTHamino acid of the terminal amino acid is liberated which leaves an intact peptide shortened by one amino acidquot Only the labeled aminoterminal residue is cleaved 3 Identify the complete amino acid sequence Repeat Edman degradation and obtain new terminal amino acids in sequence Identify each amino acid obtained via ionexchange chromatography Repeat until complete sequence of peptide is obtained label release label release etc Separation of PTHAmino Acids with HPLC Highpressure liquid chromatography HPLC can be used to identify the labeled aminoterminal residues PTHamino acid cleaved during Edman degradation Rapid and highly sensitive purification technique One cycle will last less then 60 minutes This high sensitivity of HPLC makes it feasible to analyze the sequence of a protein sample eluted from a single band of an SDSpolyacrylamide gelquot Proteins Can Be Specifically Cleaved into Small Peptides to Facilitate Analysisquot Theoretically the Edman degradation method should enable one to obtain the entire sequence of a protein Realistically speaking an impure mixture would be obtained with peptides longer than 50 residues Solution cleave proteins into smaller peptides with chemical reagents such as cyanogen bromide or proteolytic enzymes eg trypsin Table 33 lists different chemical reagents and enzymes that can be used to cleaved proteins Individual Peptides Can Be Overlapped to Obtain Primary Structure of Original Protein Once proteins are cleaved into smaller peptides and the Edman degradation method is applied the amino acid sequences of segments smaller peptides of the protein are known but the order of these segments is not yet definedquot The sequence of peptides that make up the primary structure of the original protein is obtained by quotoverlappingquot peptides and finding common sequences Disulfide Bond Reduction Proteins composed of several polypeptide chains must initially be dissociated before undergoing sequencing Disulfide bonds linking polypeptide chains are reduced with chemical agents such as betamercaptoethanol or dithiothreitol To prevent the cysteine residues from recombining and reforming disulfide bonds they separated polypeptide chains are then alkylated with iodoacetate to form stable Scarboxymethyl derivativesquot Immunology Provides Important Techniques with Which to Investigate Proteinsquot The exquisite specificity of antibodies for their target proteins provides a means to tag a specific protein so that it can be isolated quantified or visualizedquot Antibodies and Antigens Proteins can be used to function as an antigen in an organism and subsequently lead to the production of antibodies Antibodies contain antigenbinding sites on their structures The Vshaped portions of Yshaped antibody are the Fab domains that contain the antigenbinding sites at their ends The specificity of the antibodyantigen interaction is a consequence of the shape complementarity between the two surfacesquot Monoclonal vs Polyclonal Antibodies Monoclonal antibodies clones of a single antibodyproducing cell Recognize one epitope region Polyclonal antibodies heterogeneous mixture of antibodies Recognize various epitope regions Useful in detecting a protein present in low concentration The Preparation of Monoclonal Antibodies Using Mice P P rPSNNtquot 7 8 Inject antigen into mouse Obtain mouse s spleen cells several weeks later Fuse spleen cells and cellcultured myeloma cells in polyethylene glycol Select and grow hybrid cellsquot Select cells making antibody of desired specificityquot Collections of cells shown to produce the desired antibody are subdivided and reassayed This process is repeated until a pure cell line a clone producing a single antibody is isolatedquot Grow desired clones in mass culture Obtain monoclonal antibodies Proteins Can Be Detected and Quantified By Using An EnzymeLinked Immunosorbent Assay ELISAquot Indirect ELISA used for the detection of an antibody Sandwich ELISA used for the detection of an antigen Western Blotting Permits the Detection of Proteins Separated By Gel Electrophoresisquot Separate proteins on an SDSpolyacrylamide gel via gel electrophoresis Transfer proteins from gel to a polymer membrane Incubate membrane with a primary antibody specific to the protein of interest the protein acts as the antigen After washing the membrane incubate the membrane with a secondary antibody tagged with a radioactive or orescent label that is specific to the primary an body Fluorescence or radioactivity will appear as a band on a photographic film and will subsequently indicate the binding of the secondary antibody to the primary antibody and the binding of the primary antibody to the protein of interest thus enabling the detection of proteins separated by gel electrophoresis via primary and secondary antibodies Mass Spectrometry Mass spectrometry enables one to determine the mass of a molecule of interest or analyte and subsequently enables the identification of peptides and proteins Three essential components of a mass spectrometer the ion source the mass analyzer the detector Ion source converts analyte molecules into gaseous charged forms gasphase ions Mass analyzer measures the masstocharge ratio mz of the analyte ions MatrixAssisted Laser Desorption Ionization Timeof Flight MALDI TOF Steps Analyte protein sample is mixed with a matrix and evaporated until dryness The matrix is an aromatic compound that can absorb light at specific wavelengthsquot The protein sample is ionized converted into gasphase ions by a laser beam An electric field accelerates the ions through the ight tube toward the detector the lightest ions arrive firstquot A clock triggered by the laser beam measures the time of ight TOF of the gas phase ions A shorter TOF will designate a smaller ion less mass while a longer TOF will specify a larger ion greater mass Analyze peaks on a graph with the mass charge ratio on the xaxis and the intensity on the yaxis to identify molecules of interest peaks indicate molecules Tandem Mass Spectrometry Mass spectrometry that utilizes two mass analyzers Edman degradation alternative for sequencing peptides Protocol 1 Break peptides into fragments or product ions by bombarding with inert gaseous ions such as helium or argon in first mass spectrometer 2 Detect product ions in second mass analyzer The mass differences between the product ions indicate the amino acid sequence of the precursor peptide ionquot Protein Identification Through a Combination of Various Techniques Eg Analysis of nuclearpore complex from yeast Purify nuclearpore complex from yeast cells Separate identify and quantify purified complex via HPLC and subsequent gel electrophoresis Isolate bands on gel correlating to sample Cleave bands with trypsin Analyze cleaved bands with MALDITOF mass spectrometry Compare fragments obtained with amino acid sequences deduced from the DNA sequence of the yeast genomequot Utility of Synthetic Peptides quotSyn thetic peptides can serve as antigens to stimulate the formation of specific antibodies quotSyn thetic peptides can be used to isolate receptors for many hormones and other signal molecules quotSyn thetic peptides can serve as drugs quotStudying synthetic peptides can help define the rules governing the threedimensional structure of proteins Peptide Synthesis by Automated SolidPhase Methods Protect amino group with a protecting group such as a tertbutyloxycarbonyl t Boc group Protect carboxylterminal amino acid from peptide bond formation by attaching the Cterminal to a solid insoluble resin Deprotect amino terminus by removing tBoc protecting group with tri uoroacetic acid Couple the free amino terminus of resinbound amino acid with the DCC activated carboxyl group of the next amino acidquot to form a peptide bond The next amino acidquot has an unprotected free carboxyl group and a protected amino group tBoc group Repeat for addition amino acid additions Remove peptide from resin at end of synthesis with hydro uoric acid Remove protecting groups on potentially reactive side chains ThreeDimensional Protein Structure Can Be Determined by XRay Crystallography and NMR Spectroscopyquot XRay Crystallography Reveals threedimensional protein structure in atomic detailquot Components of analysis obtain a protein crystal have a source of xrays have a detector Protein crystal proteins or protein complexes to be analyzed by xray crystallography must be in a crystal form in which all protein molecules are oriented in a fixed repeated arrangement with respect to one anotherquot The addition of ammonium sulfate or some other salt to the protein may lead to the protein crystallization Principle underlying xrays Electrons scatter Xrays the amplitude of the wave scattered by an atom is proportional to its number of electrons The scattered waves recombine the scattered waves reinforce one another at the lm or detector if they are in phase there and they cancel one another ifthey are out of phase The way in which the scattered waves recombine depends only on the atomic arrangemen t Challenges It is difficult to obtain ordered crystal structures of some proteins Appropriate lenses for focusing xrays to form an image do not exist however electrondensity maps can be used to determine the structure of the proteinquot The resolution plays a role in the quality of xray analysis NMR Spectroscopy Method used for determining protein structures Based on principle that spinning charges generate a magnetic field Uses energy differences associated with transitions from a lower energy state to a higher energy state Different compounds have different frequencies or chemical shifts ppm Nuclear Overhauser Effect The nucleus of atom A interacts closely with the nucleus of atom B when in close proximity Enables the identification of neighboring atoms within chemical structures Lecture 916 Thermodynamics of Catalysis p 219229 Enzymes Enzymes are catalysts responsible for accelerating reactions Enzymes are involved in most reactions taking place in biological systems Enzymes themselves are unaffected by the reactions they catalyze Enzymes stabilize transition states Enzymes either catalyze one chemical reaction or several closely related reactionsquot Eg Proteolytic enzymes that catalyze the hydrolysis of a peptide bond also catalyze the hydrolysis of an ester bond two closely related reactions Related reactions can be useful measures of enzymatic activity Enzymes are highly specific both in the reactions that they catalyze and in their choice of reactants which are called substrates Eg Trypsin and thrombin mediate the cleavage of a particular bond only at a specific region Fig 81 The threedimensional structure of an enzyme defines its specificity Cofactors Cofactors small molecules that enable many enzymes to catalyze reactions Apoenzyme an enzyme without its cofactorquot Haloenzyme a catalytically active enzymequot Apoenzyme cofactor haloenzyme Table 82 lists common enzyme cofactors we do not need to memorize but must familiarize ourselves with Cofactors 1 Metals 2 Small organic molecules called coenzymes often derived from vitaminsquot Prosthetic groups tightly bound coenzymesquot Cosubstrates loosely associated coenzymesquot released along with substrates Enzymes Can Transform Energy From One Form Into Anotherquot Enzymes play a role in photosynthesis light energy 9 chemicalbond energy Enzymes play a role in cellular respiration free energy derived from food ATP Myosin an enzyme converts ATP mechanical energy required for muscle contraction Enzymatic pumps in cell membranes establish via ATP chemical and electrical gradients that are also different forms of energy Laws of Thermodynamics From Lecture First Law The total amount of energy within a system and its surroundings is constantquot Energy cannot be created or destroyed Second Law The total entropy S of a system and its surroundings always increases for a spontaneous processquot Reactions Assurroundings 39Aern T Assurroundings Astotal Asrxn Astotal Asrxn 39Aern T Astotal Asrxn 39 Aern T AG Aern 39 TAern TAern represents the total amount of energy absorbed by a system and therefore has units of energy S Entropy H Enthalpy T Temperature in Kelvins K G Gibbs free energy Gibbs Free Energy G Measure of useful energy Change in Free Energy AG Aern lt 0 9 Reaction is spontaneous exergonic reaction energy released Aern 0 9 System is in a state of equilibrium AH TAS Aern gt 0 9 Reaction is nonspontaneous endergonic reaction energy absorbed Aern is a state function and thus independent of the path or molecular mechanism of the reaction Aern provides no information about the rate of a reactionquot The Standard FreeEnergy Change of a Reaction Is Related to the Equilibrium Constant ABCD AG AG RT ln CD AB AG standard freeenergy change standard condition implies the reactants A B C and D being present at a concentration of 10 M R gas constant T absolute temperature in Kelvins A B C D molar concentrations of the reactants 0 AG RT ln CD 9 equilibrium AB AG RT ln CD 9 equilibrium AB K eq CD 9 under standard conditions AHB AG RT ln Km 9 equilibrium K39eq 10AG RT K39eq 10497247 9 after substituting for R and T AG expressed in kilojoules per mole K eq 1039AG 39136 9 AG expressed in kcal per mole From lecture For each 10fold change in K eq the AG changes by 136 kcalmol because AG is related to K eq by dependence on the concentration of substrates and products of the reactionquot Whether the AG for a reaction is larger smaller or the same as AG depends on the concentrations of the reactants and productsquot Reactions that are not spontaneous based on AG can be made spontaneous by adjusting the concentrations of reactants and productsquot Note 1 cal 4184 1 k 1000 1kcal 1000 cal 1k 0239 kcal Study example given on p 224 on the isomerization of DHAP to GAP Table 83 Enzymes and Equilibrium Enzymes accelerate the rate at which an equilibrium is achieved but they do not change the equilibrium of a chemical reaction The equilibrium position is a function only of the freeenergy di erence between reactants and products Enzymes Accelerate Reactions by Facilitating the Formation of the Transition Statequot S 9 X 9 P S substrate X transition state P product X 9 transition state highest free energy state state where the molecule is no longer the substrate but not quite the product leaststable AG leE Gs Gibbs free energy of activation Free energy of transition state free energy of substrate Enzymes function to lower the activation energy or in other words enzymes facilitate the formation of the transition statequot Figure 83 is important enzymes decrease the activation energy K1 v S r X 9 P K equilibrium constant for the formation of X transition state v rate of formation of product from X v is proportional to the concentration of X because only X can be converted into product X is related to the freeenergy difference AG activation energy between X and S Therefore the overall rate of reaction V depends on AGquot Lowering A695 correlates to an increase in V lowering activation energy increases the rate afformation of product quotEnzymes accelerate reactions by decreasing AG the activation energy quotThe essence of catalysis is stabilization of the transition state K V 8 9 P S and S are in equilibrium K S S The higher the barrier AG the lower S and the slower the rate of P product formation vS Enzymes decrease the activation energy AG and increase S and the formation of product vS The Formation of An EnzymeSubstrate Complex is the First Step in Enzymatic Catalysisquot Evidence For the Existence of An EnzymeSubstrate Complex The fact that an enzymecatalyzed reaction has a maximal velocity suggests the formation of a discrete enzymesubstrate complexquot The maximal velocity of the reaction indicates that all catalytic sites of an enzyme were saturated with substrate thus preventing a further increase in reaction velocity Xray crystallography Highresolution images of enzymesubstrate complexes serve as evidence Spectroscopic characteristics Spectroscopic characteristics of many enzymes and substrates change on the formation of an ES complexquot Active Site Region on an enzyme that binds substrates and cofactors Contains catalytic groups involved in the formation and breaking of bonds Responsible for enabling an enzyme to lower the activation energy of a reaction Common Features of Active Sites quotThe active site is a threedimensional cleft or crevice Generally located in interior of enzyme Formed by interactions between distant residues from different parts of the amino acid sequence sterically more favorable than adjacent residues interacting quotThe active site takes up a small part of the total volume of an enzyme The majority of the enzyme is not the active site but rather amino acids that enable the formation of the threedimensional active site quotActive sites are unique microenvironmen ts Active sites are nonpolar microenvironments water excluded unless a reactant Active sites can also be composed of polar residues these sites are exposed to water Substrates are bound to enzymes by multiple weak attractionsquot Noncovalent interactions are responsible for enzymesubstrate complexes The specificity of binding depends on the precisely defined arrangement of atoms in an active sitequot The active site must be complementary in shape to the bound substrate Lecture 918 Kinetics of Catalysis I p 229240 Kinetics Is the Study of Reaction Ratesquot The rate V of a chemical reaction A 9 P is the rate of disappearance of reactant A AAA T or the rate of the formation of product P APA T o V kA 9 Firstorder reaction units 5391 k rate constant o V kA2 9 Secondorder reaction units M39ls39l MichaelisMenten Equation Describes the kinetics rates of chemical reactions of many enzymes k k2 E S lt gt ES f E P K l k 2 k1 and k2 are analogous to k1 and kg in the equation in the textbook E enzyme S substrate P product ES enzymesubstrate complex Once the enzymesubstrate ES complex forms with rate constant k1 the complex can either dissociate back to E S with rate constant k1 or it can proceed to form product with rate constant k2 E P can reverse and reform the ES complex via rate constant k2 The ES complex is a necessary intermediate for enzyme catalysis The concentration of substrates and the concentration of product do not change at equilibrium because the conversion of substrate to product and vice versa occur at equivalent rates k1 k2 E S quotk ES E 1 1 The above equation is the MichaelisMenten Equation modified for rate of reaction at times closer to zero V0 initial velocity Because the equation depicts the reaction towards its beginning product formation is negligible and there is no reverse reaction for reforming the ES complex from the enzyme and product Continuing under the condition that we are observing early stages of the reaction V0 where product formation is negligible very low P V0 k2ES Rate of formation of ES k1E S Rate of formation of ES k1 k2ES Now assuming the reaction is at a steadystate where the concentrations of the intermediates ES stays the same even if the concentrations of starting materials and products are changingquot a k1E S k1 k2 ES b ES ES k1 k2k1 c KM k1 k2 k1 9 Michaelis constant has units of concentration Inserting c into b E5 ESl KM Now put into consideration the fact that the substrate concentration S is usually significantly greater than enzyme concentration E Furthermore the concentration of uncombined substrate S is very nearly equal to the total substrate concentrationquot E Eh ES E concentration of uncombined enzyme ET total enzyme concentration ES concentration of enzymesubstrate complex Now use this equation in ES ES KM to form ES Elr ESS KM Now solve for ES to get 135 EIIIJLS KM 1S KM OR ES ET l S KM Now using Vo k2ES V0 k2ET 1 SKM VmaX occurs when ES ET and all catalytic sites on the enzyme are saturated with substrate Vmax kzlElT The MichaelisMenten Equation V0 Vmax 1 SKM When S lt KM V0 Vmax KMS 9 first order reaction rate is directly proportional to substrate concentration S When S gtgt KM V0 Vmax 9 zero order reaction rate is independent of substrate concentration When S KM V0 VmaX 2 9 KM is equal to the substrate concentration at which the reaction rate is half its maximal valuequot Utility of the MichaelisMenten Equation From Lecture KM serves as a good approximation of S in vivoquot The fraction of sites filled can be estimatedquot KM is related to the rate constants for the individual steps in the catalytic scheme for the enzymequot The max rate provides an estimate of the turnover number of the enzyme eg the moles of substrate converted to productquot lgLat M is a Measure of Catalytic Efficiency When S gtgt KM the rate of catalysis VmaX a function of kcat This condition where enzymes are heavily saturated V0 Vmax however is less common than when most active sites of enzymes are not saturated with substrate Therefore considering the more common condition where S ltlt KM Vo L LaLlEHS KM And because S ltlt KM E is almost equal to ET V0 lcitS ET KM So when S ltlt KM the enzymatic velocity depends on lcat KM Physical Limits to the Efficiency of An Enzyme kczant KM 1La11 ank lt k1 kl kcat 1 kcat When kcat gtgt k1 the rate of production formation is fast and kcatkM approaches k1 This rate cannot be faster than the diffusioncontrolled encounter of an enzyme and its substratequot
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