Notes for Lectures 9 and 10
Notes for Lectures 9 and 10 BCM 475 - M001
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BCM 475 - M001
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This 8 page Class Notes was uploaded by Annie Notetaker on Sunday September 27, 2015. The Class Notes belongs to BCM 475 - M001 at Syracuse University taught by M. Braiman, R. Welch in Fall 2015. Since its upload, it has received 101 views. For similar materials see Biochemistry I in Biochemistry at Syracuse University.
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Lecture 921 Kinetics of Catalysis II p 240246 Physical Limits to the Efficiency of An Enzyme kcat KM kcatkl kcatk1 lt k1 k1 kcatk1 kcat Hence diffusion limits the rate of the reaction Most Biochemical Reactions Include Multiple Substratesquot A B P Q 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 1 Enzyme binds coenzyme NADH first 2 Pyruvate then binds 3 EnzymeNADHpyruvate complex forms ternary complex enzyme 2 substrates 4 Catalysis 5 EnzymeLactateNADcomplex produced ternary complex enzyme 2 products 6 Lactate is released first 7 Then NAD is released Sequential Reaction Random Sequential Mechanism Random addition of substrates and random release of products Consider reaction Creatine ATP Phosphocreatine ADP In this reaction either creatine or ATP may bind first and either phosphocreatine or ADP may be released firstquot Ternary complexes for substrates and products form as in ordered sequential mechanisms 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 enzymequot Substituted enzyme intermediates form in doubledisplacement reactions Substituted enzyme intermediates are covalently modified enzyme intermediates Consider the reaction Aspartate alphaKetoglutarate Oxaloacetate Glutamate Following the binding of the enzyme and its substrate aspartate the enzyme is covalently modified and obtains an amino group from aspartate to form enzyme NH3 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 sitesquot 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 Eg 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 substratequot Uncompetitive Inhibition The inhibitor binds only to the enzymesubstrate complexquot 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 A noncompetitive inhibitor acts by decreasing the concentration of functional enzyme rather than by diminishing the proportion of enzyme molecules that are bound to substratequot 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 EI E1 Ki Dissociation constant for enzymeinhibitor complex E Concentration of enzyme 1 Concentration of inhibitor EIConcentration 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 11 Ki KMapp KM in presence of inhibitor KM Michaelis constant the substrate concentration at which the reaction rate is half its maximal valuequot KMapp is a function of 1 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 ESI and reducing the presence of ES 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 enzymeinhibitor substrate complex does not proceed to form productquot Vmax is decreased to Vappmax KM remains the same because only the concentration of functional enzyme has decreased Vappmax Vmax 1 quot39 Il Ki Increasing substrate concentration does not a ect the inhibition Figure 818 Kinetics of noncompetitive inhibitor 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 1S 1Vmax 9Reciprocal of MM equation 1S 9 x axis 1 V0 9 yaxis slope 9KM Vmax yintercept9 1 Vmax x intercept 9 1 KM DoubleReciprocal Plots Used to distinguish between competitive uncompetitive and noncompetitive inhibitors For competitive inhibition 1Vo 1Vmax KMVmax1 llKi1SD The slope of the plot is increased by the factor 1 IKi in the presence of a competitive inhibitorquot For uncompetitive inhibition 1Vo KlemaXIS 1Vmax1 llKi The yintercept changes increases by a factor of 1 1 K1 For noncompetitive inhibition Vmaxapp Vmax 1 Ki KM remains the same Yintercept increases Slope increases Irreversible Inhibitors Can Be Used to Map the Active Sitequot 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 GroupSpecific Reagents React with specific side chains of amino acidsquot Affinity Labels Molecules that are structurally similar to the substrate for an enzyme and that covalently bind to activeside residuesquot Possess greater affinity for an enzyme s active site than that of groupspecific reagents Suicide Inhibitors Aka mechanismbased inhibitors Modified substrates that provide the most specific means for modifying an enzyme s active site Penicillin An Example of an Irreversible Inhibitor 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 placequot Penicillin also acts as a suicide inhibitor because the peptidase participates in its own inactivationquot Lecture 923 Mechanics of Catalysis I p 253262 Strategies Used by Enzymes For Catalysis Covalent Catalysis Catalysis in which a nucleophile or other reactive group within enzyme s active site temporarily covalently binds to substrate Eg Chymotrypsin s strategy of catalysis General AcidBase Catalysis A molecule other than water plays the role of a proton donor or acceptorquot Catalysis by Approximation Catalysis in which distinct substrates bind to adjacent active sites Metal Ion Catalysis Metal ions can facilitate the formation of nucleophiles serve as a bridge between enzyme and substratequot H H serve as an electrophilequot Proteases Facilitate Protein Turnover Protein turnover the balance between the formation of protein protein synthesis and the breakdown of protein protein degradation Enables the recycling of damaged or altered protein Ingested proteins must be degraded for proper absorption in the gut Proteases break peptide bonds via hydrolysis addition of water The partial double bond character present in peptide bonds accounts for the bonds resistance against proteolysis Chymotrypsin Proteolytic enzyme Cleaves peptide bonds on carboxyl end of aromatic or large hydrophobic amino acids eg tryptophan tyrosine phenylalanine methionine Conducts covalent catalysis Conducts nucleophilic attack on carbonyl carbon of substrate Nucleophile covalently binds to substrate Possesses an extremely reactive serine residue that plays a major role in catalysis Treatment of chymotrypsin with diisopropylphospho uoridate DIPF an organo uorophosphate led to the irreversible inactivation of chymotrypsin with a chemical modification only on the serine residue Figure 92 Kinetics of Chymotrypsin Catalysis The enzyme kinetics of chymotrypsin were studied by reacting chymotrypsin with an NacetylLphenylalanine pnitrophenyl ester 0 chromogenic substrate During the reaction chymotrypsin cleaves the chromogenic substrate and produces a product called pnitrophenolate Pnitrophenolate is yellow and therefore the measurements of the absorbance of light revealed the amount of pnitrophenolate being producedquot and further revealed the catalytic efficiency of chymotrypsin Kinetics for the cleavage of the chromogenic substrate under steadystate conditions KM 20 uM kcat 77 s1 The cleaving of the chromogenic substrate by chymotrypsin chymotrypsin catalysis proceeds in two steps 1 Burst phase presteady state Rapid burst of enzymatic activity The acyl group of the substrate becomes covalently attached to the enzyme as pnitrophenolate is releasedquot to form the acylenzyme intermediate 2 Steadystate phase Deacylation hydrolysis of the acylenzyme intermediate and regeneration of free enzyme Figures 94 and 95 Structure of Chymotrypsin Figure 96 Roughly spherical Contains three polypeptide chains linked by disulfide bonds It chymotrypsin is synthesized as a single polypeptide termed chymotrypsinogen which is activated by the proteolytic cleavage of the polypeptide to yield the three chainsquot The serine residue the active site is located in a cleft on the surface of chymotrypsin The structure of the active site explained the special reactivity of serine 195quot Catalytic triad Rgroup of serine 195 hydrogen bonded to imidazole ring of histidine 57 hydrogen bonded to carboxylate group of aspartate 102 The aspartate residue positions the histidine residue to become a better proton acceptor via hydrogen bonding and electrostatic effects while the histidine residue positions the serine residue to become a better proton donator by polarizing serine s hydroxyl group and hence a more effective nucleophile via formation of an alkoxide ion Asp 102 His 57 Ser 195 Figure 97 Mechanism of Peptide Hydrolysis by Chymotrypsin 1 Substrate binds to chymotrypsin The destabilization of the catalytic triad upon chemical interactions between chymotrypsin s serine residue and the substrate s peptide bond initiates peptide hydrolysis Tetrahedral intermediate formed Oxygen atom of the hydroxyl group of serine performs a nucleophilic attack on target peptide carbonyl group Histidine extracts hydrogen on serine s hydroxyl group The oxyanion hole stabilizes the tetrahedral intermediate Figure 99 Collapse of tetrahedral intermediate Acylenzyme formed upon intermediate s collapse Transfer of the proton being held by the positively charged histidine residue to the amino group formed by cleavage of the peptide bondquot Release of the amine component of substrate The first stage of peptide hydrolysis acylation by chymotrypsin is now complete Water enters active site A water molecule takes the place occupied earlier by the amine component of the substratequot Histidine functions as an acid catalyst and stabilizes a proton on water Formation of tetrahedral intermediate Water performs a nucleophilic attack on the acylenzyme intermediate Collapse of tetrahedral intermediate Formation of carboxylic acid product Release of carboxylic acid product Stabilization of negatively charged tetrahedral intermediate Origin of Specificity for Chymotrypsin Cleavage Chymotrypsin cleaves peptide bonds just past residues with large hydrophobic side chainsquot because the proteolytic enzyme possesses a deep specificity pocket lined with hydrophobic residues into which the long hydrophobic residues can favorably fit Figure 910 The specificity pocket is known as the S1 pocket Examples of long hydrophobic side chains phenylalanine tryptophan The binding of an appropriate side chain in to this packet positions the adjacent peptide bond in to the active site for cleavagequot Catalytic Triads Are Found in Other Hydrolytic Enzymesquot Enzymes trypsin and elastase contain sequences that are 40 identical to the protein sequences of chymotrypsin Trypsin cleaves peptide bond past residues with long positively charged side chains arginine lysine Elastase cleaves peptide bond past small side chains alanine serine Chymotrypsin trypsin and elastase catalyze reactions by similar mechanisms but each posses varying substrate specificity Structural differences exist in the S1 pockets of chymotrypsin trypsin and elastase differences that lead to the different substrate specificities Trypsin contains an aspartate residue at bottom of S1 pocket Elastase contains two valine residues at top of S1 pocket Figure 913 Subtilisin Protease found in bacteria Not a homolog of chymotrypsin like trypsin or elastase but still contains a catalytic triad and an oxyanion hole
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