Study Guide I
Study Guide I BCM 475 - M001
Popular in Biochemistry I
Popular in Biochemistry
This 30 page Study Guide was uploaded by Annie Notetaker on Monday September 21, 2015. The Study Guide belongs to BCM 475 - M001 at Syracuse University taught by M. Braiman, R. Welch in Fall 2015. Since its upload, it has received 441 views. For similar materials see Biochemistry I in Biochemistry at Syracuse University.
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Biochemistry The study of the chemistry that occurs within living organisms m Linear polymer of four monomers Contains a sugarphosphate sugar deoxyribose backbone with the following nitrogen containing bases 1 adenine A 2 cytosine C 3 guanine G 4 thymine T A pairs with T while G pairs with C Hydrogen bonding is responsible for holding speci c base pairs together Doublehelical structure formed when two single strands of DNA intertwine Sugarphosphate backbones located on exterior of double heliX while the bases are located inside the helical structure Covalent Bonds Strongest bonds formed by the sharing of electrons between two atoms E g CC 9 covalent bond with a bond length of 154 A formed between two carbon atoms Note 1A 103910 m 10398 cm 01 nm Resonance structures display different patterns of covalent bonding e g electron pushing within a benzene ring Noncovalent Bonds Weaker than covalent bonds Four types of noncovalent bonds 1 Electrostatic interactions 2 Hydrogen bonds 3 Van der Waals interactions 4 Hydrophobic interactions l Electrostatic Interactions Attractive interactions between a cation and an anion E Elia Dr E energy K proportionality constant q1 charge on atom 1 q2 charge on atom 2 D dielectric constant r distance between two atoms A An attractive force tween to molecules results in a E value 2 Hydrogen Bonds Fundamentally an electrostatic interaction Weaker than covalent bonds Form when a hydrogen bonded to an electronegative atom N O or F is attracted to a nearby electronegative atom NH hydrogen39bond donor N hydrogenbond acceptor Partial negative charges on both N atoms Partial positive charge on H because the electronegative N atom is pulling electron density away from the H atom Properties of Water Water is a polar molecule Bent structure Asymmetric distribution of charge Water is highly cohesive Water molecules interact with other water molecules through hydrogen bonds Eg structure of ice 3 Van der Waals Interactions Aka dipole induced dipole interactions or London forces Based on principle that electrons within an atom are not symmetrically distributed with time thus creating in one point in time one end that is electron rich partial negative charge and another end that is electron poor partial positive charge Van der Waals interactions occur when the asymmetry in electronic distribution in one atom induces the same asymmetric electronic charge in an adjacent or nearby atoms9 dipole inducing dipole The energy of van der Waals interactions increases as the distance between two atoms decreases below the van der Waals contact distance repulsion Van der Waals contact distance the distance between two atoms where the energy of the interactions is most favorable 4 Hydrophobic interactions Nonpolar molecules interact more often with other nonpolar molecules when placed in a solution of water When a nonpolar molecule is placed in water water molecules form a network via hydrogen bonds around the nonpolar molecule When two nonpolar groups aggregate some water molecules that had initially been in the network around the nonpolar molecules are released resulting in a favorable event Double Helix The structure of DNA that displays the four types of noncovalent bonding First Law of Thermodynamics The total energy of a system and its surrounding is constant Energy is neither created nor destroyed Second Law of Thermodynamics The total entropy of a system plus that of its surroundings always increases Entropy S degree of disorder in a system AG AHsystem39TASsystem G Gibbs free energy H Enthalpy T Temperature in kelvins K S Entropy AcidBase Reactions pH logH1 pOH logOH39 pHpOH 14 Kw HOH39 10 x 103914 KW ionic product of water Ka H A39 HA HA acid H cation hydrogen ion Aquot anion pKa log K Ka acid dissociation constant PH PKa 10gA39lHA Henders0n Hasselbalch equation Buffers Responsible for regulating pH Resist changes in pH Molecular Representations Spacefilling models Ballandstick models Structural formulas Properties of Proteins Proteins are linear polymers built of monomer units called amino acids Proteins are formed by linking the ucarboxyl group of one amino acid to the uamino group of another amino acid Proteins contain a Wide range of functional groups Eg alcohols thiols thioethers carboxylic acids carboxamides basic groups Proteins can interact with one another and with other biological macromolecules to form complex assemblies Some proteins are quite rigid Whereas others display a considerable exibility E g rigid proteins in the cytoskeleton or in connective tissue and exible proteins in hinges spring or levers Amino Acids Building blocks of proteins Consist of the following parts central carbon atom 0t carbon amino group carboxylic group hydrogen atom R group the side chain 0t amino acids are chiral and may exist either as a L isomer or a D isomer L and D isomers are mirror images n isomer l isomer Only L amino acids are found in proteins The L isomer has S absolute con guration counterclockwise progression from the highest priority substituent to the lowest priority substituent based on atomic number Amino acids as Dipolar Ions Amino acids eXist as dipolar ions at neutral pH or near physiological pH Dipolar ions zwitterions Protonated amino group NH3 Deprotonated carboxyl group COO39 Ionization State of Amino Acids In acid solution protonated amino group NH3 and protonated carboxyl group COOH In neutral pH zwitterion form predominates with a protonated amino group NH3 and deprotonated carboxyl group COO39 In basic solution deprotonated amino group NH2 and deprotonated carboxyl group COO Classi cation of Amino Acids Hydrophobic amino acids with nonpolar R groups Polar amino acids with neutral R groups but unevenly distributed charge Positively charged amino acids with R groups that have a positive charge at physiological pH Negatively charged amino acids with R groups that have a negative charge at physiological pH Small Hydrophobic Amino Acids Glycine Gly G Achiral Contains two H atoms bonded to the Qt carbon Alanine Ala A R group methyl group CH3 Large Hydrophobic Amino Acids Valine Val V Leucine Leu L Isoleucine He 1 Contains a side chain with another chiral center Methionine Met M 4 Hydrophobic Rigid Amino Acids Proline Pro P R group covalently bonded to the nitrogen and 0t carbon Aromatic Amino Acids Phenylalanine Phe F R group phenyl ring Purely hydrophobic Tyrosine Tyr Y Tryptophan Trp W Not as hydrophobic as phenylalanine because of NH groups Polar Uncharged Amino Acids with OH Groups Attached to Hydrophobic Side Chain Serine Ser S Threonine Thr T Tyrosine Tyr Y Hydroxyl groups make these three amino acids more hydrophilic Polar Uncharged Amino Acids with Carboxamide Groups Asparagine Asn N Glutamine Gln Q Polar ThiolContaining Reactive Amino Acid Cysteine Cys C Forms disul de bonds with other cysteine amino acides upon oxidation Polar Hydrophilic Positively Charged Amino Acids Lysine Lys K Arginine Arg R Histidine His H Two possible forms at neutral pH Hydrophilic Negatively Charged Amino Acids Aspartate Asp D Glutamate Glu E Primary Structure Amino Acid Sequence of Proteins Proteins are linear polymers formed by linking the ucarboxyl group of one amino acid to the uamino group of another amino acid with a peptide or amide bond The formation of a peptide bond is accompanied by the loss of a water molecule hydrolysis Energy input required for forming peptide bonds Rate of hydrolysis slow Polypeptide Chain Amino acids linked by peptide bonds Components constant polypeptide backbone main chain of carbonyl and NH groups variable side chains with distinctive R groups Residue each amino acid unit in a polypeptide Has polarity Beginning of polypeptide chain aminoterminal residue Nterminal End of polypeptide chain carboxylterminal residue Cterminal Most natural polypeptide chains contain between 502000 amino acid residues and are commonly referred to as proteins Protein mass 5500220000 g mol391 MW of amino acid residue 110 g mol39l Note one Dalton one atomic mass unit 50000 g mol391 50000 Daltons 50 kilo Daltons Crosslinked Linear Polypeptide Chain E g two cysteine residues linked by a disulfide bond via oxidation reactions Cystine two linked cysteines Geometry of the Protein Backbone Peptide bonds are planar Resonance structures restrict atoms to a plane For two amino acids linked by a peptide bond siX atoms CXamino acid 1 Camino acid 1 Oamiiic acid 1 Namino acid 2 H amino acid 2 and Ca amino acid 2 are located in the same plane Doublebond character within polypeptide chains leads to conformational restriction Trans and Cis Peptide Bonds Con gurations for planar peptide bonds cis two ucarbons on the same side of bond and trans two ucarbons on opposite side of bond Almost all peptide bonds in proteins are trans to reduce steric hindrances Both trans and cis conformations present in XPro bonds Sterics present in both configurations Rotational Flexibility in Polypeptide Chains Single bonds eXist between the amino group and the ucarbon atom and between the OL carbon atom and the carbonyl group Free rotation possible about these single bonds Free rotation enables various protein folding Torsion Angle Aka Dihedral angles Measure of the rotation about a bond Phi 1 the angle of rotation about the bond between the nitrogen and the ucarbon atoms Psi w the angle of rotation about the bond between the ucarbon and the carbonyl carbon atoms Ramachandran Diagrams Twodimensional plot displaying which combinations of phi and psi angles are possible and which combinations are not possible due to steric hindrances Secondary Structure of Proteins Polypeptide chains can fold into regular structures such as the alpha helix the beta sheet and turns and loops The Alpha Helix Tightlycoiled rodlike structure with the polypeptide backbone forming the interior region and side chains extending outward to prevent steric clashes Stabilized by interchain hydrogen bonding between NH and CO groups The CO group of residue i forms a hydrogen bond with the NH group of residue i 4 Screw Sense The direction in which a helical structure rotates with respect to its axis Both righthanded clockwise or lefthanded counterclockwise helices occur All 0L helices found in proteins will be righthanded helices due to decreased steric clash Beta Sheets Two or more adjacent beta strands two or more polypeptide chains linked via hydrogen bonding Antiparallel residues aligned in two chains or parallel residues offset in two chains H R H O H R H o C I n X I II N cN CNCCN C Iquotgt J39I Ii R 91 Ii R I 11 Ixquot parallel H B it o H R H 5 C I II x I II II 39 Iii 3 E R IlIRR anti uj 1 F 3 ILII H parallel II I 39I ll 39 C CNCCNCX CNxCCN lt pf 39139 6 1391 R a lt3939gt r39 Mixed beta sheets can contain both antiparallel and parallel sheets Beta sheets can obtain a at structure but most display a slightly twisted shape Fatty acidbinding protein rich in beta sheets Polypeptide chains can change direction by making reverse turns and loops Reverse turn beta turn or hairpin turn the CO group of residue i of a polypeptide is hydrogen bonded to the NH group of residue i 3 and stabilizes the turn Omega loops structure that enables polypeptide chains to change their directions Do not possess regular structure but are structurally rigid Both reverse turns and omega loops lie on the surface of the protein structure Tertiary Protein Structure The threedimensional arrangement of a polypeptide chain Myglobin Serves as an example of a protein possessing a tertiary structure 7 Compact oxygenbinding protein responsible for providing oxygen to muscle cells Single polypeptide chain of 153 amino acids Contains a heme group nonpolypeptide prosthetic group that buries into the protein structure in an aqueous environment The interior of the protein consists almost entirely of nonpolar residues such as leucine valine methionine and phenylalanine While the exterior of the protein contains both polar and nonpolar residues F or thermodynamic stability watersoluble polypeptide chains fold in a manner such that the hydrophobic side chains are clustered in the interior of the protein to avoid contact with the polar hydrophilic aqueous environment whereas polar charged chains are exposed on the surface Membrane Proteins Membrane proteins differ from soluble proteins in the distribution of hydrophobic and hydrophilic groups Integral membrane proteins extend through the lipid bilayer end to end Peripheral membrane proteins do not span the entire lipid bilayer Can be fastened to the lipid bilayer membrane via hydrophobic amino acids Electrostatic interactions and hydrogen bonding With lipid head groups are responsible for the binding of peripheral membrane proteins to the membrane Bacteriorhodopsin vs Bacterial Porin Bacteriorhodopsin Membrane protein Consists of seven membranespanning alphahelices Membranespanning alphahelices are the most common structural motif in membrane proteins Consists of mostly nonpolar uncharged amino acid residues that are in contact With the lipid bilayer s hydrocarbon core or With other alphahelices Bacterial Porin Bacterial membrane protein Materfillcd han Largely hydrophobic hydrophilic c nel exterior Porin has a hydrophobic exterior With charged polar hydrophilic chains in its interior R groups towards exterior facing lipid bilayer are hydrophobic While R groups towards interior facing water channel are hydrophilic Betabarrel structure consists entirely of B strands The reason for this exceptional structure of porin is the fact that porin functions in a hydrophobic environment Whereas soluble proteins such as myoglobin function in a hydrophilic aqueous solution Prostaglandin Hg Integral membrane protein The parts of the protein that interact with the hydrophobic parts of the membrane are coated with nonpolar amino acid side chains Whereas those parts that interact with the aqueous environment are much more hydrophilic Transmembrane Helices Can Be Accurately Predicted From Amino Acid Sequences Membranespanning alphahelices mostly nonpolar uncharged amino acid residues are the most common structural motif in membrane proteins The hydrocarbon core of a membrane is typically 30A wide a length that can be traversed by an alpha helix consisting of 20 amino acid residues The free energy changes that accompany the transfer of a alphahelical segment amino acid residues from a hydrophobic membrane environment to a hydrophilic aqueous environment can be used to determine potential transmembrane helices amino acid residues of a alpha helix that are located Within the membrane bilayer A hydropathy plot indicates potential transmembrane helices Includes free energy values for the transfer of 20 amino acid residues of an alphahelix from a hydrophobic membrane environment to a hydrophilic aqueous environment Peaks greater than or equal to 84 kJ mol391 on a hydropathy plot are indicative of a potential transmembrane helix E g a peak greater than 84 kJ mol391 in a hydropathy plot for glycophorin indicates the single alpha helix indicative of a transmembrane helix Proteins Consist of Structurally Independent Domains Many polypeptide chains fold into domains Domains are compact globular units with sizes ranging from 30 to 400 amino acid residues Domains are connected with exible linkers Quaternary Structure of Proteins Multisubunit Structure The spatial arrangement of subunits and the nature of their interactions Subunit l polypeptide chain in a protein that may be identical to or different from other subunits Within a protein A dimer is a quaternary structure with 2 identical subunits Hemoglobin Quaternary structure Serves as an example of a protein composed of different subunits Contains 2 identical alpha subunits 2 identical beta subunits to form a uz z tetramer Rhinovirus Protein Coat Quaternary structure Contains 60 copies of four distinct subunits that collectively become a spherical shell The Relationship Between the Amino Acid Sequence of a Protein and Its ThreeDimensional Structure Ribonuclease enzyme composed of 124 amino acid residues crosslinked by four disulfide bonds The following experiment with ribonuclease revealed how the amino acid sequence of a protein determines the threedimensional structure of the protein 1 The threedimensional structure of ribonuclease was destroyed with 8 M urea Chemical agents such as urea or guanidinium chloride disrupt secondary tertiary and quaternary structures of proteins Most polypeptide chains devoid of crosslinks assume a randomcoil conformation in 8M urea or 6M guanidinium chloride 2 Disulf1de bonds within the enzyme were reduced with Bmercaptoethanol Disulf1des cystines converted to free sulfhydryls cysteines Treatment with Bmercaptoethanol in 8 M urea resulted in a fully reduced randomly coiled peptide without its normal activity denatured 3 Bmercaptoethanol and urea removed by dialysis Urea removed before Bmercaptoethanol Removal of the two chemical agents responsible for denaturing the enzyme led to the oxidation of sulfhydryl groups by air the reformation of the enzyme into its active conformation and the restoration of enzymatic activity Conclusion the information needed to speci the catalytically active structure of ribonuclease is contained in its amino acid sequence Protein Folding Neighboring proteins play a role in stabilizing a particular peptide conformation Protein Folding and Unfolding is a Highly Cooperative Process Protein folding and unfolding is an all or none process that results from a cooperative transition Proteins cannot directly jump from a folded state to an unfolded state or vice versa Unstable shortlived intermediate structures form between folded states and unfolded states Proteins Fold By Progressive Stabilization of Intermediates Rather Than By Random Search The freeenergy difference between the folded and the unfolded states of a typical 100 residue protein is 42 kJ mol39l and thus each residue contributes on average only 042 kJ mol391 of energy to maintain the folded state Fig 26 Depicts how the thermodynamics of protein folding resembles a funnel Some Proteins are Inherently Unstructured and Can Exist in Multiple Conformations lntrinsically unstructured proteins IUPs have no distinct structure and therefore vary structurally based on interactions with other proteins Lymphotactin exists in two conformation 10 Proteins Fold By Progressive Stabilization of Intermediates Rather Than By Random Search The freeenergy difference between the folded and the unfolded states of a typical 100 residue protein is 42 kJ mol39l and thus each residue contributes on average only 042 kJ mol391 of energy to maintain the folded state Fig 26 Depicts how the thermodynamics of protein folding resembles a funnel Some Proteins are Inherently Unstructured and Can Exist in Multiple Conformations Intrinsically unstructured proteins lUPs have no distinct structure and therefore vary structurally based on interactions with other proteins Lymphotactin eXists in two conformation The Proteome the Functional Representation of the Genome directly from lecture As of 2015 there are about 50000 genomes sequenced 22500 of Eukaryotes and 475000 for prokaryotes The human genome contains about 3 billion base pairs of DNA and about 23000 genes A genome represents a list of all the genes that make up an organism but it does not tell you when those genes are made into proteins or how and when they interact with each other The proteome of an organism signi es a more complex level of information content encompassing the types functions and interactions of proteins within its biological environment Protein Purification Key to understanding protein function Enables the determination of amino acid sequences the protein s biochemical function and the protein s structure Protein Purification Requires an Assay Assay a test that will enable one to determine the presence of the protein of interest by identifying one of its unique properties Increased assay specificity increased puri cation effectiveness E g Enzyme lactate dehydrogenase catalyzes the reaction of lactate NAD to pyruvate NADH H An assay can be used to measure the enzyme activity an enzyme s ability to effectively catalyze a reaction and lower the activation energy of lactate dehydrogenase NADH absorbs light at 340 nm and NAD does not absorb light at this wavelength Therefore an assay can be utilized to determine how much of a sample absorbs light after a certain amount of time to subsequently determine the activity of lactate dehydrogenase increased absorption of light means the enzyme is present and working because its job is to catalyze the reaction and lead to the production of lightabsorbing NADH 11 Protein Puri cation Requires the Determination of the Concentration of Protein in the Sample Being Assayed Speci c activity Enzyme activity Protein concentration The overall goal of protein puri cation is to maximize the speci c activity Proteins Must Be Released From the Cell to be Puri ed To release the proteins from the cells the cell initially undergoes homogenization The homogenate then undergoes d erential centrifugation to yield several fractions of decreasing density each still containing hundreds of different proteins From Fig 31 we can see how during differential centrifugation pellets of nuclear fractions form faster than pellets for mitochondrial fractions while pellets for mitochondrial fractions form faster than pellets for microsomal fractions Finally each fraction is assayed and subsequently puri ed Proteins Can Be Puri ed According to Solubility Size Charge and Binding Af nity Separation by Solubility Salting out Utilizes the principle that most proteins are less soluble at high salt concentrations Separation by Size Dialysis Performed by placing a protein mixture concentrated solution into a dialysis bag with a semipermeable membrane and subsequently submerging the bag into a buffer solution Smaller molecules and ions diffuse out of the dialysis bag while larger molecules and protein aggregates remain inside the bag Not an effective technique for purifying proteins GelFiltration Chromatography Aka molecular exclusion chromatography Utilizes a column containing porous beads While small molecules can insert themselves inside the beads and in the solution between the beads large molecules can only ow to the aqueous solution between the beads Large molecules ow more rapidly through this column and emerge rst because a smaller volume is accessible to them This puri cation technique is good for separating samples with a 50 difference in size Longer columns will enable the separation of samples with increased differences Separation by Charge Ionexchange chromatography Negatively charged beads placed in a column will attract and bind proteins with a net positive charge at pH 7 opposite charges attract while negatively charged molecules will simply ow through the column Proteins bound to the negatively charged beads are eluted by increasing the salt concentration of the eluting buffer Sodium ions compete with positively charged groups on the protein for binding to the column 12 Positively charged proteins can be separated by chromatography on negatively charged carboxymethylcellulose CMcelluose columns Negatively charged proteins can be separated by anion exchange on positively charged diethylaminoethylcellulose DEAEcellulose columns Af nity Chromatography Highly selective puri cation technique A substrate molecule with high af nity selectivity for a protein of interest is covalently attached to a column When sample is added to the column the substrate will selectively bind the protein of interest A wash buffer will then be poured through the column to remove unbound proteins Desired protein can be eluted off the column by the addition of a high concentration of a soluble form of the substrate molecule with high speci city for the protein of interest Gel Electrophoresis Method for separating proteins based on their molecular mass Electrophoresis the phenomenon in which a molecule with a net charge will move in an electric eld v Ezf v velocity of migration of a protein or any molecule in an electric eld E electric eld strength 2 net charge on the protein f frictional coef cient Before gel electrophoresis proteins are initially dissolved in sodium dodecyl sulfate SDS a detergent to denature the proteins and to give the proteins an overall negative charge The overall negative charge on the proteins will then enable them to migrate to the positive pole when an electric eld is applied during gel electrophoresis Polyacrylamide gels are commonly used for gel electrophoresis Polyacrylamide gels can have varying pore sizes Gels with a higher concentration of acrylamide are used for larger molecules The electrophoretic mobility of many proteins in SDSpolyacrylamide gels is inversely proportional to the logarithm of their mass An exception to this relationship occurs with carbohydraterich proteins and membrane proteins and some phosphorylated proteins For SDSpolyacrylamide gel electrophoresis proteins that differ in mass by about 2 eg 50 and 5 lkd arising from a difference of about 10 amino acids can usually be distinguished Isoelectric Focusing Isoelectric point pl pH at which the net charge is zero and the point where electrophoretic mobility is zero Puri cation technique used to separate proteins based on their isoelectric points in the absence of SDS A pH gradient is rst established in the gel The gel is then run enabling proteins to migrate to the region on the gel where the pH of the gel is equal to the pl of the protein 13 TwoDimensional Electrophoresis The combination of isoelectric focusing with SDSPAGE Ultracentrifugation Technique used to purify proteins s m 1 V p f s sedimentation coef cient m mass of the particle V bar partial speci c volume p density of the medium f frictional coef cient 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 Speci c amino acid sequences in many proteins serve as signals designating their destinations or controlling their processing 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 pro les for the amino acids The amount of each amino acid present is determined from the absorbance e g on sulfonated polystyrene resin acidic amino acids will ow out of column rst 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 N terrninal 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 acid Only the labeled aminoterminal residue is cleaved 3 Identify the complete amino acid sequence 14 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 amino terminal residues PTHamino acid cleaved during Edman degradation Rapid and highly sensitive puri cation 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 gel Proteins Can Be Speci cally Cleaved into Small Peptides to Facilitate Analysis 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 e g 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 defined The sequence of peptides that make up the primary structure of the original protein is obtained by overlapping peptides and finding common sequences Disulf1de Bond Reduction Proteins composed of several polypeptide chains must initially be dissociated before undergoing sequencing Disulf1de 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 S carboxymethyl derivatives Immunology Provides Important Techniques with Which to Investigate Proteins The exquisite specificity of antibodies for their target proteins provides a means to tag a specific protein so that it can be isolated quantif1ed or visualized Antibodies and Antigens Proteins can be used to function as an antigen in an organism and subsequently lead to the production of antibodies Antibody immunoglobulin Ig protein Antibodies are produced in response to an antigen a substance the body recognizes as foreign 15 Eg of antigens foreign proteins polysaccharides nucleic acids synthetic peptides attached to a macromolecular carrier Antibodies elicit immune responses in response to recognizing the epitope region of an antigen a speci c group or cluster of amino acids on the target molecule the antigen Antibodies contain antigenbinding sites on their structures Antibodies are Yshaped proteins structurally composed of four chains two identical heavy chains and two identical light chains linked by disul de bonds and noncovalent interactions The V shaped portions of Yshaped antibody are the Fab domains that contain the antigenbinding sites at their ends The lower Ishaped portion of Yshaped antibodies is the Fc domain a domain composed of two heavy chains The Fab domains are linked to the Fc domain by exible linkers The speci city of the antibodyantigen interaction is a consequence of the shape complementarity between the two surfaces 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 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 cells Select cells making antibody of desired speci city 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 isolated 7 Grow desired clones in mass culture 8 Obtain monoclonal antibodies 9959959 Proteins Can Be Detected and Quanti ed By Using An EnzymeLinked Immunosorbent Assay ELISA 1 Indirect ELISA used for the detection of an antibody 1 Coat bottom of a well with antigens 2 Add primary antibodies to well only antibodies speci c to bound antigens will bind to the antigens adsorbed to the bottom of the well 3 Add enzymelinked secondary antibodies that have high af nity for primary antibodies 4 Add substrate that will enable the enzyme linked to the secondary antibody to catalyze a reaction and produce a colored product 5 Analyze the rate of color formation that is proportional to the amount of antibody originally present Indirect ELISAs are commonly used to test blood samples for an HIV infection The antigens used to coat the bottom of a well are antigens recognized only by antibodies 16 generated in HIVpositive patients Therefore the formation of a colored product following the addition of the substrate indicates that the secondary enzymelinked antibody recognized the primary antibody present only in blood of HIVpositive patient that had in turn recognized the adsorbed antigen Sandwich ELISA used for the detection of an antigen Coat bottom of a well with monoclonal antibodies speci c to antigen of interest Add solution containing antigen into well Speci c antigens bind to adsorbed antibodies Add enzymelinked secondary monoclonal antibody speci c to antigens Add substrate and analyze the rate of color formation that is proportional to the amount of antigen present MPWF C Western Blotting Permits the Detection of Proteins Separated By Gel Electrophoresis Separate proteins on an SDSpolyacrylamide gel via gel electrophoresis Transfer proteins from gel to a polymer membrane Incubate membrane with a primary antibody speci c 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 speci c to the primary antibody Fluorescence or radioactivity will appear as a band on a photographic lm 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 Fluorescent Markers Make the Visualization of Proteins in the Cell Possible Cells can be stained with uorescencelabeled antibodies and examined by uorescence microscopy to reveal the location of a protein of interest 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 TimeofFlight 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 speci c wavelengths The protein sample is ionized converted into gasphase ions by a laser beam An electric eld accelerates the ions through the ight tube toward the detector the lightest ions arrive rst A clock triggered by the laser beam measures the time of ight TOP of the gasphase 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 masscharge ratio on the xaxis and the intensity on the yaxis to identify molecules of interest peaks indicate molecules 17 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 rst 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 ion Protein Identification Through a Combination of Various Techniques E g 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 genome Utility of Synthetic Peptides Synthetic peptides can serve as antigens to stimulate the formation of speci c antibodies Synthetic peptides can be used to isolate receptors for many hormones and other signal molecules Synthetic peptides can serve as drugs Studying 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 tBoc group Protect carboxylterminal amino acid from peptide bond formation by attaching the C terminal 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 DCCactivated carboxyl group of the next amino acid to form a peptide bond The next amino acid 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 Spectroscopy 18 XRay Crystallography Reveals threedimensional protein structure in atomic detail 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 xed repeated arrangement with respect to one another 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 if they are out of phase The way in which the scattered waves recombine depends only on the atomic arrangement 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 protein 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 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 reactions E g 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 19 E g Trypsin and thrombin mediate the cleavage of a particular bond only at a speci c region Fig 81 The threedimensional structure of an enzyme de nes its speci city Cofactors Cofactors small molecules that enable many enzymes to catalyze reactions Apoenzyme an enzyme without its cofactor Haloenzyme a catalytically active enzyme Apoenzyme cofactor haloenzyme Table 82 lists common enzyme cofactors we do not need to memorize but must familiarize ourselves with Cofactors l Metals 2 Small organic molecules called coenzymes often derived from vitamins Prosthetic groups tightly bound coenzymes Cosubstrates loosely associated coenzymes released along with substrates Enzymes Can Transform Energy From One Form Into Another Enzymes play a role in photosynthesis light energy 9 chemicalbond energy Enzymes play a role in cellular respiration free energy derived from food9 ATP Myosin an enzyme converts ATP9 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 constant Energy cannot be created or destroyed Second Law The total entropy S of a system and its surroundings always increases for a spontaneous process Reactions ASsurroundings 39Aern T ASsurroundings Astotal Aern Astotal Aern 2 39Aern T AStotal Aern 39 Aern T 0 AG AHm1 TASm1 TASrxn 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 AGrxn lt 0 9 Reaction is spontaneous exergonic reaction energy released AGrxn 0 9 System is in a state of equilibrium AH TAS AGrxn gt 0 9 Reaction is nonspontaneous endergonic reaction energy absorbed 20 AGrxn is a state function and thus independent of the path or molecular mechanism of the reaction AGrxn provides no information about the rate of a reaction The Standard FreeEnergy Change of a Reaction ls Related to the Equilibrium Constant ABCD AG AG RT ln CD A 13 AG0 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 BCD molar concentrations of the reactants 0 AG RT ln CD 9 equilibrium AllBl AG RT ln CD 9 equilibrium AllBl K eq CD 9 under standard conditions A B AG RT ln K eq 9 equilibrium K 1 oAGO IRT eq AG 247 K 10 eq 9 after substituting for R and T AG expressed in kiloj oules per mole K eq 1039AGO I 13936 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 reaction Whether the AG for a reaction is larger smaller or the same as AG depends on the concentrations of the reactants and products Reactions that are not spontaneous based on AG can be made spontaneous by adjusting the concentrations of reactants and products Note 1 cal 4184 J 1 kJ 1000J 1kcal 1000 cal 1kJ 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 eeenergy di erence between reactants and products 21 Enzymes Accelerate Reactions by Facilitating the Formation of the Transition State s 9 xi 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 AG11E GX 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 state Figure 83 is important enzymes decrease the activation energy K V S 2 xi 9 P Ki 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 X11E is related to the freeenergy difference AG activation energy between X11 and S Therefore the overall rate of reaction V depends on AGZ LoweringAGf correlates to an increase in V lowering activation energy increases the rate of formation of product Enzymes accelerate reactions by decreasing AGI the activation energy The essence of catalysis is stabilization of the transition state K V s s1 9 P S and S are in equilibrium K S S The higher the barrier AG1 the lower Si and the slower the rate of P product formation vSZ Enzymes decrease the activation energy AG1 and increase Si and the formation of product vS The Formation of An EnzymeSubstrate Complex is the First Step in Enzymatic Catalysis 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 complex 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 complex 22 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 The active Site is a threedimensional cleft 0r 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 The active Site takes up a small part 0fthe 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 Active Sites are unique microenvironments 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 attractions Noncovalent interactions are responsible for enzymesubstrate complexes The speci city of binding depends on the precisely defined arrangement of atoms in an active site The active site must be complementary in shape to the bound substrate Kinetics Is the Study of Reaction Rates 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 V kA 9 Firstorder reaction units s39l k rate constant 0 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 23 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 ES quot ES FEa P kl The above equation is the MichaelisMenten Equation modi ed 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 kzlESl Rate of formation of ES k1ES 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 changing 3 k1ElSl k1 k2 ES b ESlEsl 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 concentration E EIT 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 ElT ESDlSl KM Now solve for ES to get ES 1s1 KM 24 OR ES ElT I l SKM Now using V0 k2 ES V0 kzlElT IE1 SKM VmaX occurs when ES ET and all catalytic sites on the enzyme are saturated with substrate Vmax k2 The MichaelisMenten Equation V0 Vmax I l SKM When S lt KM V0 Vmax KMS 9 rst 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 value Utility of the MichaelisMenten Equation From Lecture KM serves as a good approximation of S in vivo The fraction of sites lled can be estimated KM is related to the rate constants for the individual steps in the catalytic scheme for the enzyme The maX rate provides an estimate of the turnover number of the enzyme e g the moles of substrate converted to product per mole of enzyme when the enzyme is fully saturated with substrate IELatQM is a Measure of Catalytic Ef ciency 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 V0 LicatEl 5 KM And because S ltlt KM E is almost equal to ET V0 1ltcatSl ElT KM So when S ltlt KM the enzymatic velocity depends on 15 KM 25 Physical Limits to the Ef ciency of An Enzyme kcat IM kcatl1 EEK lt k1 k1 kcat kl kcat When kcat gtgt k4 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 substrate in aqueous solutions the diffusion limit is between 108 and 109 1 1 S M 3 Physical Limits to the Ef ciency of An Enzyme kcat IM kcatl li EEK lt k1 k1 kcat kl kcat Hence diffusion limits the rate of the reaction Most Biochemical Reactions Include Multiple Substrates A B P Q 9 The general formula of an enzymatic reaction A and B are two substrates or a substrate and its cofactor P and Q are two products Multiple Substrate Reactions Two classes 1 Sequential reactions 2 Doubledisplacement reactions Sequential reactions requires the complete formation of enzymesubstrate complexes before the formation of a product Sequential Reaction Ordered Sequential Mechanism Occurs for many enzymes with NAD or NADH substrates Consider reaction Pyruvate NADH H Lactate NAD Steps of this ordered sequential mechanism Enzyme binds coenzyme NADH first Pyruvate then binds EnzymeNADHpyruvate complex forms 9 ternary complex enzyme 2 substrates Catalysis EnzymeLactateNAD complex produced 9 ternary complex enzyme 2 products Lactate is released first Then NAD is released 999 9 Sequential ReactionRandom Sequential Mechanism Random addition of substrates and random release of products Consider reaction Creatine ATP 2 Phosphocreatine ADP In this reaction either creatine or ATP may bind first and either phosphocreatine or ADP may be released first Ternary complexes for substrates and products form as in ordered sequential mechanisms 26 DoubleDisplacement PingPong Reactions The release of products acts independently of the binding of substrates One or more products are released before all substrates bind the enzyme Substituted enzyme intermediates form in doubledisplacement reactions Substituted enzyme intermediates are covalently modi ed enzyme intermediates Consider the reaction Aspartate alphaKetoglutarate 2 Oxaloacetate Glutamate Following the binding of the enzyme and its substrate aspartate the enzyme is covalently modi ed and obtains an amino group from aspartate to form enzymeNH3 EnzymeNH3 is the substituted enzyme intermediate Allosteric Enzymes Composed of multiple subunits and multiple active sites Fail to obey MichaelisMenten kinetics Display sigmoidal activation kinetics Cooperative binding of substrate is detected in allosteric enzymes The binding of substrate to one active site facilitates the binding of substrate to the other active sites Figure 813 Different Types of Enzyme Inhibition Irreversible Inhibition The tight binding covalent or noncovalent of an inhibitor to an enzyme that increases the time during which the enzyme is inhibited from its normal catalytic activity Slow dissociation of the inhibitor from the enzyme E g Penicillin and aspirin are beneficial irreversible inhibitors both drugs covalently modify their target enzymes Reversible Inhibition The inhibitor is not bound to its target enzyme for an extended period of time Rapid dissociation of inhibitor from enzyme Types of reversible inhibition 1 Competitive inhibition 2 Uncompetitive inhibition 3 Noncompetitive inhibition Competitive Inhibition The binding of an inhibitor to an enzyme s active site and subsequent prevention of the substrate from binding to the enzyme s active site The enzyme can only bind either the substrate or the inhibitor Because the enzyme cannot bind both the substrate and the inhibitor competition between the substrate and inhibitor occurs A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate Uncompetitive Inhibition The inhibitor binds only to the enzymesubstrate complex Increasing substrate concentration does not affect the inhibition Noncompetitive Inhibition Inhibition in which the inhibitor can bind either to an enzyme free of substrate or an enzymesubstrate compleX 27 A noncompetitive inhibitor acts by decreasing the concentration of functional enzyme rather than by diminishing the proportion of enzyme molecules that are bound to substrate Increasing substrate concentration does not affect the inhibition Mixed Inhibition Inhibition in which the inhibitor acts both as a competitive and noncompetitive inhibitor Kinetics of Competitive Inhibition Both the inhibitor and the substrate compete for the enzyme s active site in competitive inhibition Ki Ellll E1 Ki 9 Dissociation constant for enzymeinhibitor complex B 9 Concentration of enzyme I 9 Concentration of inhibitor EI9 Concentration of enzymeinhibitor complex Smaller Ki 9 greater inhibition less inhibitor dissociating from enzymeinhibitor complex A significant increase in S can overcome competitive inhibition Fig 816 Kinetics of a competitive inhibitor KMapp KM 1 Il Ki KMapp 9 KM in presence of inhibitor KM 9 Michaelis constant the substrate concentration at which the reaction rate is half its maximal value KMapp is a function of I Kinetics of Uncompetitive Inhibition Inhibition in which the uncompetitive inhibitor I binds only to an enzymesubstrate complex ES to form ESI Increasing uncompetitive inhibitor I leads to a lower VmaX because the complex ESI formed does not lead to the formation of product Uncompetitive inhibitor I lowers KM by increasing the formation of E81 and reducing the presence of BS Fig 817 Kinetics of an uncompetitive inhibitor Kinetics of Noncompetitive Inhibition In the presence of a noncompetitive inhibitor on an enzyme a substrate is still capable of binding to the active site of the enzyme however the enzymeinhibitorsubstrate complex does not proceed to form product VmaX is decreased to Vappmax KM remains the same because only the concentration of functional enzyme has decreased VM 1 I K Increasing substrate concentration does not a ect the inhibition Figure 818 Kinetics of noncompetitive inhibitor Vappmax 28 LineweaverBurk Plot Figure 812 learn to understand intercepts Doublereciprocal plot This plot is obtained by taking the reciprocal of both sides of the MichaelisMenten equation so as to obtain a straightline plot the MM equation generates a hyperbolic curve V0 Vmax S 9 MichaelisMenten equation SKM 1V0 KMVmax X US 1Vmax 9 Reciprocal of MM equation lS 9 XaXis 1 V0 9 yaXis slope 9 KM VmaX yintercept 9 l VmaX Xintercept 9 1 KM DoubleReciprocal Plots Used to distinguish between competitive uncompetitive and noncompetitive inhibitors For competitive inhibition 1V0 1Vmax KMVmax IHi1S The slope of the plot is increased by the factor 1 IKi in the presence of a competitive inhibitor For uncompetitive inhibition 1V0 KMVmaXS 1Vmax1 I i The yintercept changes increases by a factor of l IKi For noncompetitive inhibition Vmaxapp Vmax KM remains the same Yintercept increases Slope increases Irreversible Inhibitors Can Be Used to Map the Active Site The tight covalent bond certain irreversible inhibitors form with enzymes enables one to determine the enzyme s functional groups responsible for its catalytic activity By covalently binding to enzymes irreversible inhibitors modify the enzyme s functional groups and subsequently enable the identification of the functional groups Irreversible inhibitors 1 Groupspecific reagents 2 Reactive substrate analogs affinity labels 3 Suicide inhibitors 29 GroupSpeci c Reagents React with speci c side chains of amino acids Af nity Labels Molecules that are structurally similar to the substrate for an enzyme and that covalently bind to activeside residues Possess greater af nity for an enzyme s active site than that of groupspeci c reagents Suicide Inhibitors Aka mechanismbased inhibitors Modi ed substrates that provide the most speci c means for modifying an enzyme s active site Penicillin An Example of an Irreversible Inhibitor Note Irreversible inhibitor suicide inhibitor Antibiotic Glycopeptide transpeptidase is an enzyme known to accelerate the formation of cross links responsible for supporting and strengthening the peptidoglycan linear polysaccharide chains linked by peptides present in the cell wall of the bacterium S aureus Under normal conditions transpeptidase catalyzes the formation of an acyl intermediate with the Dalanine residue of its normal substrate Penicillin however mimics the Dalanine residue acts like the substrate and covalently binds to the active site of transpeptidase Penicillin is therefore acting as an irreversible inhibitor In conclusion the transpeptidase is irreversibly inhibited and cellwall synthesis of bacteria cannot take place Penicillin also acts as a suicide inhibitor because the peptidase participates in its own inactivation Quotations indicate text obtained directly from textbook References Berg Jeremy John Tymoczko and Lubert Stryer Biochemistry 7th ed WH Freeman 2012 l 246 Print 30
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