Notes for Lectures 11-13
Notes for Lectures 11-13 BCM 475 - M001
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BCM 475 - M001
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Lecture 928 Mechanics of Catalysis II p 262284 Peptide Hydrolysis By Chymotrypsin Figure 98 Oxyanion hole Stabilizes the tetrahedral intermediate Keeps water away Stabilizes the negative charge on the oxygen atom Subtilisin Protease found in bacteria Not a homolog of Chymotrypsin like trypsin or elastase but still contains a catalytic triad and an oxyanion hole Figure 914 Subtilisin possesses a catalytic triad consisting of aspartic acid 32 histidine 64 and serine 22 1 Sitedirected mutagenesis has been performed to determine the importance of subtilisin s catalytic triad and oxyanion hole Residues of the catalytic triad were mutated to alanine and the activity of the mutated enzyme was measuredquot Result KM unchanged substrate binding normal kcat reduced for each residue A decrease in enzymatic activity decreased kcat in response to the mutations indicated the importance of the catalytic triad to subtilisin s enzymatic activity Proteases Function to cleave proteins by hydrolysis the addition of water to a peptide bond Cysteine proteases Aspartyl proteases Metalloproteases Cystein Protease Utilizes a histidineactivated cysteine residue as the nucleophile attacking the carbonyl carbon atom of the target peptide bond Similar to mechanism of Chymotrypsin during peptide hydrolysis but differs in that cysteine proteases due to their sulfur atom functioning as a better nucleophile than serine s oxygen atom in Chymotrypsin does not require a full catalytic triad for enzymatic activity Eg papain Aspartyl Protease Utilizes an aspartateactivated water molecule as the nucleophile attacking the carbonyl carbon atom of the target peptide bond A deprotonated aspartic acid residue activates a water molecule by attracting a proton on the water molecule with its negatively charged oxygen atom A protonated aspartic acid residue polarizes the carbonyl group on the target peptide bond with its proton the carbonyl carbon now has a greater partial positive charge and is therefore more prone to attack by a nucleophile In conclusion the water molecule activated by the deprotonated aspartic acid residue attacks the target carbonyl carbon atom polarized by the protonated aspartic acid residue Eg renin pepsin both possess twofold symmetry HIV protease Dimer of identical subunits The enzyme may have originally existed as separate subunitsquot Metalloprotease Utilizes a metalactivated water molecule as the nucleophile attacking the carbonyl carbon atom of the target peptide bond The metal is bound to the active site of the enzyme and is most of the time zinc Eg thermolysin carboxypeptidase A 9 zinc proteases Common Features of Active Sites of Cysteine Aspartyl and Metalloproteases The active site includes features that act to activate a water molecule or another nucleophilequot The active site includes features that act to polarize the peptide carbonyl groupquot The active site includes features that act to stabilize a tetrahedral intermediatequot Figure 917 Protease Inhibitors Function as Drugs Captopril Inhibits angiotensinconverting enzyme metalloprotease Regulates blood pressure Indinavir Crixivan and Retrovir Inhibits HIV protease an aspartyl protease that cleaves multidomain viral proteins into their active formsquot HIV protease is a dimer of identical subunitsquot Indinavir resembles the peptide substrate of the HIV proteasequot Figures 918920 A Fast Reaction k1 C02 H20 2 Carbonic acid Bicarbonate ion HC0339 H k1 This hydration reaction occurs at a fast rate even in the absence of a catalyst At neutral pH k1 00027 M391 s1 In water k1 015 s1 k1 50 5391 K1 54 X 105 C02 H2C03 3401 Carbonic anhydrases increase the rate of this fast reaction even more km 106 s1 for most active carbonic anhydrases Properties of Metal Ions Responsible for Increasing Chemical Reactivity Positively charged Ability to form strong yet kinetically labile bondsquot Capacity to be stable in more than one oxidation statequot Carbonic Anhydrase Contains bound zinc ion essential for the enzyme s catalytic activity Carbonic anhydrase functions optimally at high pH Zinc Zinc is found only in the 2 state in biological systemsquot Zinc site of carbonic anhydrase 11 Located in cleft at center of carbonic anhydrase The zinc ion is bound to the imidazole rings of three histidine molecules The zinc ion is also bound to a water molecule Figure 921 Zinc is always bound to four or more ligandsquot The Hydration of Carbon Dioxide via Carbonic Anhydrase 1 The positively charged zinc ion bound to carbonic anhydrase attracts the oxygen atom of a water molecule partial negative charge The water molecule binds to the zinc center of carbonic anhydrase reducing the pKa ofwater from 157 to 7 A proton is released from the bound water molecule A zincbound hydroxide ion that will serve as a potent nucleophile is generated 2 The carbon dioxide substrate binds to the enzyme s active site and is positioned to react with the hydroxide ionquot A hydrophobic region of carbonic anhydrase next to the zinc site serves as a carbon dioxide binding site 3 The zincbound hydroxide ion conducts a nucleophilic attack on carbon dioxide Carbon dioxide is converted into bicarbonate ion HC0339 4 The bicarbonate ion is displaced from carbonic anhydrase by water Catalytic site is regenerated Testing the Zincbound Hydroxide Mechanism With a synthetic analog model system A ligand capable of binding zinc was synthesized to mimic carbonic anhydrase The zinc complex of this synthetic ligand accelerates the hydration of carbon dioxide more than lOOfold under appropriate conditionquot This result strengthened the importance of the zincbound hydroxide mechanism in carbon dioxide hydration via carbonic anhydrase A Paradox In the first step of the hydration of carbon dioxide by carbonic anhydrase the water molecule bound to the zinc ion releases a proton and generates a zincbound hydroxide ion that will later function as a potent nucleophile The rate of the reverse reaction k4 the protonation of the zincbound hydroxide ion is limited by the rate of proton diffusion k1quot Rate of proton diffusion k1 103911 M391 s1 Rate constant of backward reaction k1 must be less than or equal to 1011 M391 s1 Equilibrium constant K k1 k1 9 k1 K k1 With k1 being less than or equal to 1011 M391 s1 and K being 107 M pKa 7 k19 less than or equal to 104 s1 The rate of proton diffusion limits the rate of proton release to less than 104 s1 for a group with pKa 7 However if carbon dioxide is hydrated at a rate of 106 s1 than every step in the mechanism must take place at least this fastquot Solution Presence of a buffer Buffers can be present at concentrations above the concentration limit of protons and hydroxide ions 10397 M Buffers enable the enzyme to achieve its high catalytic ratesquot Histidine 64 of carbonic anhydrase 11 functions as a proton shuttle to enable large buffers to take part in catalytic activity The proton shuttle controls the proton transfers from and to the active sitequot Histidine 64 abstracts a proton from water bound to the zincsite of the protease while the buffer removes the abstracted proton Restriction Enzymes Aka restriction endonucleases Restriction enzymes are used to degrade viral DNA injected into host cells via viruses Bacteria and Achaea use restriction enzymes as a defense mechanism against viral infections Restriction enzymes identify recognition sequences or recognition sites in target DNA Cleave specific recognition sites Type II restriction enzymes Most well studied class of restriction enzymes Cleave DNA within their recognition sites while other restriction enzymes cleave at sites not adjacent to recognition sequences Restriction enzymes require two levels of specificity They must not degrade host DNA containing the recognition sequencesquot They must cleave only DNA molecules that contain recognition sites cognate DNA without cleaving DNA molecules that lack these sitesquot Lecture 930 Mechanics of Catalysis III p 262284 Viral vs Host DNA Similarity common recognition site Difference host DNA is methylated Result restriction enzyme fails to recognize and cleave host DNA because of the methyl groups on the host DNA The Hydrolysis of a DNA Phosphodiester Bond Catalyzed by a restriction enzyme The reaction in which the single bond connecting the 3 oxygen atom and the phosphorus atom of a DNA molecule is broken is catalyzed by a restriction endonuclease Possible mechanisms for the hydrolysis of a phosphodiester bond Covalent in termediate formation for cysteine proteases 1 Nucleophilic residue of restriction endonuclease attacks phosphate group of target DNA 2 Covalent intermediate enzyme bound to phosphate group formed 3 Hydrolysis of covalent intermediate 4 Formation of products DNA strands with a free 3 hydroxyl group and a 5 phosphoryl group The overall stereochemical configuration is retained in this mechanism because the stereochemical configuration at the phosphorus atom would be inverted and then inverted againquot during the two displacement reactions that are taking place at the phosphorus atom Two step process mirror image of mirror image is product Direct hydrolysis for metalloproteases 1 Activation of water molecule 2 Activated water molecule attacks the phosphorus atom of the target DNA 3 Breakage of phosphodiester bond The overall stereochemical configuration is inverted because a single displacement reaction occurs at the phosphorus atom One step process mirror image is product Both mechanisms covalent intermediate formation and direct hydrolysis occur by inline displacement Inline displacement The nucleophile attacking the phosphorus atom is aligned with the leaving group in the pentacoordinate transition state that is formed in the process of the hydrolysis of a phosphodiester bond Stereochemical configuration at the phosphorous atom is inverted with a single displacement reaction The Steps for Observing Stereochemical Changes that Occur During the Cleavage of a Phosphodiester Bond by a EcoRV Endonuclease One the replacement of one oxygen atom bound to the phosphorus atom of DNA with a sulfur atom to form a phosphorothioate This step enables one to more easily detect stereochemical changes that occur about the phosphorus atom during the hydrolysis of a phosphodiester bond because there are no longer two identical oxygen atoms attached to phosphorous Two the synthesis of an appropriate substrate for EcoRV containing phosphorothioates at the sites of cleavagequot Three reaction performed in 180 enriched water The location of the 18O label with respect to the sulfur atom indicates whether the reaction proceeds with inversion or retention of stereochemistryquot EcoRV cleaves the phosphodiester bond between the T and the A at the center of the recognition sequence 5 GATATC3 quot Conclusion The stereochemical con guration at the phosphorus atom was inverted only once with cleavagequot Magnesium Required particularly by restriction enzymes attacking phosphatecontaining substrates for catalysis Helps activate a water molecule for hydrolysis Positions nucleophilic water molecule for attack of phosphorus of DNA The six coordination positions of magnesium 2 with Asp COO39 3 with H20 1 with the phosphate O The Specificity of Restriction Endonucleases The unanswered question is how do restriction enzymes recognize and cleave only a specific DNA sequence containing the recognition site aka the cognate DNA The answer lies in the three dimensional structure of both the restriction endonuclease and the recognition sequence Both restriction enzymes and the sites they recognize possess twofold rotational symmetry a complex of EcoRV and DNA fragments display this twofold rotational symmetry The recognition sites are inverted repeats an arrangement that gives the three dimensional structure of the recognition site a twofold rotational symmetryquot while restriction enzymes are dimers whose two subunits are related by two fold rotational symmetryquot The corresponding symmetry between the restriction enzyme and the recognition sequence ensures specificity How Restriction Endonucleases Cleave Only Cognate DNA Sequences Although EcoRV cleaves only cognate DNA the enzyme binds to both cognate and noncognate sequences 5 GATATC3 is the recognition site for EcoRV Upon binding of the enzyme to this recognition DNA sequence the DNA sequence is distorted A kink is formed in the center of the complex due to two TA base pairs in the recognition sequence TAAT pairs are more easily distorted due to fewer hydrogen bonds when compared to CC CG pairs This distorted configuration in the cognate DNA positions the complex restriction endonuclease cognate DNA close enough to the magnesium ionbinding site for subsequent catalysis In contrast noncognate DNA does not distort as significantly as cognate DNA upon binding of the restriction enzyme and is therefore unable to bind to active site magnesium ions the DNA backbone of noncognate DNA is too far from the enzyme to complete the magnesium ionbinding sitesquot and form a complete catalytic complex The binding energy of EcoRV to cognate DNA is greater than the binding energy of the endonuclease to noncognate DNA The greater energy for the binding of EcoRV to cognate DNA is used to drive the formation of distortions and subsequently catalysis The Protection of HostCell DNA The methylation of specific adenine bases in host DNA via methylases prevents the cleavage of host DNA identical to recognition sequences in viral DNA Endonucleases do not cleave methylated recognition sites The methylation of adenine blocks the formation of hydrogen bonds between EcoRV endonuclease and cognate DNA molecules and prevents their hydrolysisquot Type II Restriction Enzymes Have a Catalytic Core in Common and Are Probably Related by Horizontal Gene Transferquot Figure 943 type II restriction enzymes have a conserved structural core and are therefore evolutionarily related Myosin Enzyme responsible for catalyzing the hydrolysis of ATP H20 9 ADP and P1 The energy associated with this thermodynamically favorable reactionquot is utilized by myosin for muscle cell movement Present in all eukaryotes Human genome more than 40 different myosins Structure elongated with globular ATPase domains responsible for ATP hydrolysis ATPase Domain MyosinATP Complex Structure Single globular domain about 750 amino acids with nucleotidebinding site at center Active site contains ATP nucleotide bound to Mg2 Magnesium ion is not a part of the active site Metal ionnucleotide complex true substrate for enzyme enzymes are only active with metal ion Dissociation constant for ATP Mg2 complex 01 mM Given that intracellular MgZ concentrations are typically in the millimolar range essentially all nucleoside triphosphates are present as N TP M92 MyosinATP Complex Stable Structural conformation prevents complex from catalysis mechanisms Conformational change necessary for complex to catalyze reactions The catalytically competent conformation of the myosin ATPase domain must bind and stabilize the transition state of the reactionquot Myosin ATPase transitionstate analog pentacoordinate transition state Due to the innate instability of the pentacoordinate intermediate structure based on phosphorus experimental observation of the structure is difficult As a solution the transition state is treated with ADP and vanadate V0430 to create a more stable transitionstate analog In the transition state analog the vanadium atom is coordinated to five oxygen atoms including one oxygen atom from ADP diametrically opposite an oxygen atom that is analogous to the attacking water molecule in the transition statequot the magnesium ion is coordinated to one oxygen atom from the vanadate one oxygen atom from the ADP two hydroxyl groups from the enzyme and two water moleculesquot The Ser 236 residue on the myosin ATPase transition state analog is positioned so as to activate the water molecule for attack of ATP A TP serves as a base to promote its own hydrolysisquot Myosin Conformational Changes Myosin undergoes a conformational change when its ATPase domain is bound to ADPvanadate instead of ATP ATPase domain complexed with ATP a region of amino acids important for catalytic function approaches the ATP nucleotide by about 2 angstroms ATPase domain complexed with ADPvandate a region of amino acids at the C terminus essential for catalytic function approach ADPvandate by nearly 25 angstroms The effect of this motion greater displacement is amplified even more as this C terminus domain is connected to other structures within the elongated structures typical of myosin moleculesquot The Altered Conformation of Myosin Persists for a Substantial Period of Timequot Myosins function at slow rates Lecture 1002 Allosteric Proteins I p 289297 Regulation of Enzymatic Activity Allosteric control The control of enzymatic activity via allosteric proteins that contain distinct regulatory sites and multiple functional sitesquot Allosteric enzymes display cooperative binding where the binding of substrate to one active site affects the activity of other functional sites Eg of an allosteric protein is aspartate transcarbamoylase ATCase Multiple forms of enzymes Isozymes isoenzymes9 homologous enzymes of a common organism catalyze identical reactions vary structurally and possess different KM and Vmax values Isozymes provide an avenue for varying regulation of the same reaction at distinct locations or times to meet the specific physiological needs in the particular tissue at a particular timequot Reversible covalent modification The control of enzymatic activity by the covalent attachment of a modifying group most commonly a phosphoryl groupquot to alter catalytic properties Proteolytic Activation The control of enzymatic activity by reversibly or irreversibly altering the inactive state of an enzyme to its active state Controlling the amount of enzyme present The control of enzymatic activity through the adjustment of enzyme concentration Aspartate Transcarbamoylase ATCase is Inhibited by CTP ATCase Carbamoyl phosphate aspartate Ncarbamoylaspartate Pi 9 cytidine triphosphate CTP ATCaseinitiated pathway ATCase is regulated by feedback inhibition the inhibition of an enzyme by the end product of the pathwayquot Low concentrations of CTP correlate to higher rates of Ncarbamoylaspartate formation but an increasing concentration of CTP leads to a decreased rate of N carbamoylaspartate formation because CTP inhibits ATCase from catalyzing the formation of Ncarbamoylaspartate to ensure subsequent intermediates in the pathway are not needlessly formed when pyrimidines are abundantquot CTP Allosteric inhibitor Binds to distinct site on ATCase Catalytic and Regulatory Subunits of ATCase Steps for separating ATCase into its different subunits 1 Treat ATCase with a mercurial compound eg phydroxymercuribenzoate that will react with sulfhydryl groups 2 Ultracentrifuge 3 Separate subunits by ionexchange chromatography or by centrifugation in a sucrose density gradient 4 Obtain sedimentation coefficients Native enzyme 9 116 S Subunit 1 9 28 S 9 regulatory r subunit 9 can bind CTP possesses no catalytic activity Subunit 2 9 58 S 9 catalytic c subunit 9 unresponsive to CTP The fully active enzyme can be reconstructed via 2 C 3 1 2 9 C6l 6 Allosteric Interactions in ATCase are Mediated by Large Changes in Quaternary Structure Structure of ATCase Two catalytic trimers stacked one on top of the other linked by three dimers of the regulatory chainsquot NphosphonacetylL aspartate PALA Bisubstrate analog analog of two substrates Used to identify the substratebinding site of ATCase Competitive inhibitor of ATCase PALA binds at sites lying at the boundaries between pairs of c chains within a catalytic trimer each catalytic trimer contributes three active sites to the complete enzymequot Upon binding of PALA to ATCase the two catalytic subunits move 12 angstroms apart and rotate 10 degrees about axis of symmetry The regulatory dimers rotate 15 degrees about axis of symmetry Different states of ATCase Tense T state 9 substrate or substrate analogs absent Relaxed R state 9 substrate or substrate analogs present Effect of adding substrate The chances of finding enzymesubstrate complex increases More enzymes in R state Homotropic e ect the effects of substrates on allosteric enzymesquot Binding of substrate stabilizes the R state The binding of CTP to the regulatory subunits of ATCase stabilizes the T state CTP binds to the regulatory subunits at a site which does not interact with the catalytic subunitsquot ATCase exists in an equilibrium between the T and R state The position of the equilibrium depends on the number of active sites that are occupied by substratequot Concerted mechanism Explains allosteric mechanism of ATCase The entire enzyme is converted from T into R affecting all catalytic sites equallyquot Sequential model The binding of ligand to one site on the complex can affect neighboring sites without causing all subunits to undergo the T to R transitionquot Allosterically regulated enzymes do not follow MichaelisMenten MM kinetics Figure 1010 ATCase leads to a sigmoidal curve Vmax reached faster that is a combination of an MM curve for the T state and another MM curve for the R state High KM T state Low KM R state Increased substrate concentration leads to an increased amount of ATCase in R state Allosteric Modulators CTP inhibits ATCase ATCase is in T state when bound to CTP CTP stabilizes the T state and subsequently lowers the probability of ATCase transitioning from the T state to the R state ATP Allosteric effector of ATCase Increases reaction rate Stabilizes the R state ATP competes with CTP for binding to regulatory sitesquot Consequently high levels of ATP prevent CTP from inhibiting the enzymequot Increased ATP Excess purine Increased ATCase activity Production of pyrimidine for balance Increased ATP energy available for mRNA synthesis and DNA replication pyrimidine synthesis Quantitative description of concerted model L T R L equilibrium constant T tense state R relaxed state For the CTPsaturated form the value of L increases from 200 to 1250quot MonodWymanChangeux Model Directly from lecture Sequential models can also account for allosteric effectsquot Sequential changes can take place in neighboring subunits without promoting a transition encompassing the entire enzyme quot Sequential models can also account for negative cooperativity for example the binding of s to one subunit decreases the affinity of other sites for squot Isozymes Homologous enzymes of a common organism Catalyze identical reactions Vary structurally Differ kinetically quotThe existence of isozymes permits the netuning of metabolism to meet the needs of a given tissue or developmental state M4 isozyme Expressed in skeletal muscle Functions best under anaerobic conditions Lower affinity for substrate H4 isozyme Expressed in heart muscle Functions best under aerobic conditions Higher affinity for substrate Note Quotations indicate text obtained directly from textbook References Berg Jeremy John Tymoczko and Lubert Stryer Biochemistry 7th ed WH Freeman 2012 l 246 Print
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