Study Guide II
Study Guide II BCM 475 - M001
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Study Guide 11 Lectures 1019 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 NacetylL phenylalanine 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 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 10 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 11 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 12 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 13 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 14 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 Lecture 105 Allosteric Proteins 11 p 297302 Enzyme Regulation Via Covalent Modification Covalent Modification Modification of enzymatic activity by covalently attaching a molecule to an enzyme Eg phosphorylation dephosphorylation acetylation Histone activity is covalently modified on lysine residues Via acetylation Reversible process in most cases Irreversible process in some other cases The irreversible attachment of a lipid group causes some proteins in signal transduction pathways such as Ras a GTPase and Src a protein tyrosine kinase to become affixed to the cytoplasmic face of the plasma membranequot Table 101 lists common types of covalent modifications Protein Phosphorylation 30 of eukaryotic proteins are phosphorylated Catalyzed by protein kinases ATP most common donor of phosphoryl groupsquot The terminal phosphoryl group of ATP is transferred to a specific amino acid of the acceptor protein or enzymequot Eukaryotic residues that are common phosphorylation sites 9 1 Serine 2 Threonine 3 Tyrosine Kinases and Phosphatases Control the Extent of Protein Phosphorylationquot Protein Kinases Catalyze protein phosphorylation Vary in degree of specificity Dedicated protein kinases phosphorylate a single protein or several closely related onesquot Multifunctional protein kinases Capable of phosphorylating more than one protein 15 Generally recognize a consensus sequence The consensus sequence of the multifunctional protein kinase is the following Arg ArgXSerZ or ArgArgXThrZ Ser or Thr9 Phosphorylation site X9 small residue Z9 large hydrophobic residue The primary determinant of specificity is the amino acid sequence surrounding the serine or threonine phosphorylation sitequot Residues a considerable distance apart however can also play a role in specificity Protein phosphatases Catalyze protein dephosphorylation Break bonds attached to phosphoryl groups via hydrolysis mechanisms Generate the reformation of original unmodified serine threonine or tyrosine residue orthophosphate Pi is also formed Phosphorylation and dephosphorylation are not reverse reactions Each is essentially irreversible under physiological conditionsquot Both phosphorylation and dephosphorylation require protein kinases and phosphatases respectively for function because both modifications occur at negligible rates in the absence of enzymesquot Phosphorylation and Dephosphorylation Reactions Both reactions are only possible in the presence of an enzyme kinase or phosphatase and a hydroxylcontaining acceptor residue serine threonine or tyrosine residue Phosphorylation occurs at the expense of ATP Dephosphorylation results in the release of P The rate of cycling between the phosphorylated and dephosphorylated states depends on the relative activities of kinases and phosphatasesquot Reasons Phosphorylation is an Effective Covalent Modification of Protein Activity 1 The free energy of phosphorylation is large Both phosphorylation and dephosphorylation are energetically favorable G 50 kImol 12 kcalmol 9 free energy provided by ATP for phosphorylation A freeenergy change of 569 kImol 136 kcalmol corresponds to a factor of 10 in an equilibrium constantquot quotA phosphoryl group adds two negative charges to a modified protein Enables the formation of new electrostatic interactions quotA phosphoryl group can form three or more hydrogen bonds quotPhosphorylation and dephosphorylation can take place in less than a second or over a span of hours Adjustable kinetics quotPhosphorylation often evokes highly amplified ejfects An activated kinase protein can in turn phosphorylate many other proteins quotA TP is the cellular energy currency 16 The use of this compound ATP as a phosphorylgroup donor links the energy status of the cell to the regulation of metabolismquot The Activation of Protein Kinase A PKA via Cyclic AMP CAMP Epinephrine adrenaline triggers 9 cAMP formation 9 cAMP 9 PKA activated by cAMP Most effects of cAMP in eukaryotic cells are achieved through the activation by cAMP of PKA Structure of PKA Two different subunits 49kd regulatory R subunit and 38 kd catalytic c subunit Inactive form cAMP absent R2C2 complex Active form bound cAMP one R2 subunit and two catalytically active C subunits Process by which cAMP activates PKA 1 Inactive PKA complex R2C2 complex contains pseudosubstrate sequence of inhibitor in catalytic sites of catalytic subunits C The pseudosubstrate sequence prevents substrate from binding to the kinase s catalytic site of the catalytic subunit 2 cAMP binds to cAMPbinding domains present on regulatory subunits 3 Pseudosubstrate sequences are allosterically removed from the catalytic sites of the catalytic subunits 4 Catalytically active subunits without a pseudosubstrate sequence present at their active sites are now able to bind substrate 5 Phosphorylation free to occur Structure of Catalytic Subunit of PKA Bound to Pseudosubstrate Inhibitor Two lobes small and large Smaller lobe9 interacts with ATPMg2 Larger lobe9 binds peptide ATP and part of the inhibitor fill a deep cleft between the lobesquot Both ATP and the inhibitor bind to the active site of PKA The PKA structure has broad significance because residues 40280 constitute a conserved catalytic core that is common to essentially all known protein kinasesquot Binding of Pseudosubstrate to PKA Pseudosubstrate sequence ArgArgAsnAlaIle Two arginine side chains of the pseudosubstrate form salt bridges with three glutamate carboxylate groupsquot The isoleucine residue of the pseudosubstrate is in contact with a pair of leucine residues of the enzymequot hydrophobic interactions 17 Lecture 107 Enzyme Regulation p 302313 Enzyme Activation by Proteolytic Cleavage Proper threedimensional protein folding is essential for many enzymes to obtain their enzymatic activity Other enzymes however additionally require the cleavage of one or more peptide bonds proteolysis for full enzymatic activity Proteolytic activation Zymogen proenzyme inactive enzyme in its threedimensional forms Cleavage does not require ATP Proteolytic activation in contrast with allosteric control and reversible covalent modification takes place just once in the life of an enzyme moleculequot and is an irreversible mechanism Cases Instances of Proteolytic Activation 1 Digestive enzymes Many digestive enzymes initially exist as zymogens 2 Blood clotting Mediated by a cascade of proteolytic activations that ensures a rapid and amplified response to traumaquot 3 Protein Hormones Insulin is derived from proinsulin by proteolytic removal of a peptidequot 4 Fibrous protein Collagen the major constituent of skin and bone is derived from procollagen a soluble precursorquot 5 Developmental processes Many developmental process such as the metamorphosis of a tadpole into a frog occur by the activation of an inactive precursor 6 Apoptosis Programmed cell death mediated by proteolytic enzymes called caspases which are synthesized in precursor form as procaspasesquot Chymotrypsinogen Inactive precursor of chymotrypsin Chymotrypsin digestive enzyme that breaks down proteins in the small intestine Synthesized in the acinar cells of the pancreas Stored in secretory granules in pancrease Chymotrypsinogen stored in secretory granules are released into the duodenum of the small intestine in response to a hormone or nerve impulse Single polypeptide chain of 245 amino acid residues Proteolytic Activation of Chymotrypsinogen 1 Chymotrypsinogen inactive enzyme is cleaved into residues 115 and residues 16245 by trypsin Trypsin cleaves the peptide bond between Arg 15 and Ile 16 18 2 nChymotrypsin active formed 3 quotnChymotrypsin then acts on other nchymotrypsin molecules by removing two dipeptides to yield alphachymotrypsin the stable form of the enzymequot Conformational Changes Accompanying the Proteolysis of Chymotrypsinogen is Responsible for the Enzymatic Activity of Chymotrypsin The newly formed aminoterminal group of isoleucine 16 turns inward and forms an ionic bond with aspartate 194 in the interior of the chymotrypsin moleculequot Formation of substratespecificity site for aromatic and bulky nonpolar groups Methionine 192 moves closer to surface Distance between residues 187 and 193 increases This cavity for binding part of the substrate is not fully formed in the zymogenquot The oxyanion hole formed during peptide hydrolysis by chymotrypsin stabilizes the tetrahedral intermediate of the reaction The oxyanion hole is incomplete in the zymogen chymotrypsinogenquot Generation of Trypsin from Trypsinogen Trypsinogen Inactive precursor of trypsin Trypsin Proteolytic enzyme Significant conformational changes in four regions of polypeptide chains about 15 of the entire molecule are observed in the activation of trypsinogen In zymogen exible regions In active form trypsin9 well defined regions The oxyanion hole generated in trypsinogen does not stabilize the tetrahedral intermediate Trypsin Common activator of all the pancreatic zymogens trypsinogen chymotrypsinogen proelastase procarboxypeptidase and prolipasequot Produced when enteropeptidase hydrolyzes a specific lysineisoleucine bond in trypsinogen Cells lining the duodenum secrete enteropeptidase The formation of trypsin by en teropeptidase is the master activation stepquot Inhibitors of Proteolytic Enzymes Zymogen inactive 9 Active proteolytic enzyme Occurs by the cleavage of a single peptide bond Irreversible process Because the generation of a protease from a zymogen by the cleavage of a single peptide bond is an irreversible process the activity must be modulated by specific inhibitors Eg Pancreatic trypsin inhibitor Molecular weight 6 kd Inhibits trypsin by binding very tightly to its active sitequot 19 Dissociation constant 01 pM Standard free energy of binding 75 kImol 18 kcalmol 8M urea or 6M guanidine hydrochloride will not denature this protein Very stable complex The inhibitor experiences significant structural change upon binding to the active site of the enzyme trypsin In essence the inhibitor is a substrate but its intrinsic structure is so nicely complementary to the enzyme s active site that it binds very tightly rarely progressing to the transition state and is turned over slowlyquot Crucial for biological processes because trypsin being an activator of other inactive enzymes could start a detrimental cascade al Antitrypsin Aka alantiproteinase Molecular weight 53 kd Protects tissues from digestion by elastasequot Elastase9 protease Functions as an antielastase Inhibits elastase by binding to the enzyme s active site by an irreversible mechanism 11 Antitrypsin deficiency mutation leads to the destruction of alveolar walls in the lungs by elastase Emphysema destructive lung disease Blood Clotting Formed in response to a signal cascade of zymogen activations Pathways leading to the initiation of blood clots 1 Intrinsic pathway This pathway is activated by exposure of anionic surfaces on rupture of the endothelial lining of the blood vesselsquot Hageman factor activated 2 Extrinsic pathway This pathway is initiated when trauma exposes tissue factor TF an integral membrane glycoproteinquot Once the tissue factor is released thrombin is generated Thrombin proteolytic enzyme Thrombin subsequently begins a signal cascade that leads to the activation of more thrombin The extrinsic and intrinsic pathways converge on a common sequence of nal steps to form a clot composed of the protein brinquot Figure 1026 Bloodclotting cascade 20 Formation of a Fibrin Clot Final step in bloodclotting cascade Thrombin Fibrinogen 9 Fibrin Structure of fibrinogen 340 kd Three globular units connected by two rodsquot Rod regions a helical coiled coils Six chains two Aa chains two BB chains and two y chains Thrombin cleaves four arginineglycine peptide bonds in the central globular region of brinogenquot The process of the formation of a fibrin clot 1 quotThrombin cleaves fibrinopeptidesA and B from the central globule of brinogenquot 2 Two A peptides of individual lengths of 18 residues are released from each Aa chain Two B peptides of individual lengths of 20 residues are released from each BB chain 3 A fibrin monomer am2 is generated9 fibrinogen molecule without fibrinopeptides A and B peptides 4 GlyHis Arg sequences are generated at the amino terminal end of the beta chains upon cleavage of B peptides GlyProArg sequences are generated at the amino terminal end of the alpha chains upon cleavage of A peptides 5 GlyProArg sequences of alpha chains interact with gamma subunits of a different monomer 9 polymerization 6 GlyHis Arg sequences of beta chains interact with beta subunits of a different monomer 9 polymerization 7 Formation of fibrin clot The newly formed quotsoft clot is stabilized by the formation of amide bonds between the side chains of lysine and glutamine residues in di ferent monomersquot Fibrin Orderly fibrous arrays formed with the coming together of fibrin monomers generated through the cleavage of fibrinogen via thrombin Electron Micrograph of Fibrin Fibrin has a periodic structure that repeats every 23 nmquot along the fiber axis Prothrombin Zymogen of thrombin Structure of prothrombin Amino terminus Gla domain ycarboxyglutamaterich domain9 Kringle domain9 Kringle domain9 Serine protease domain Carboxyl terminus Prothrombin inactive 9 Thrombin active 1 Proteolytic cleavage of the bond between arginine 274 and threonine 275 to release a fragment containing the first three domainsquot 2 Cleavage of the bond between arginine 323 and isoleucine 324quot 21 3 Generation of thrombin from prothrombin Vitamin K Essential for the synthesis of prothrombin and several other clotting factorsquot Deficiency defective blood koagulation Vitamin K antagonists Dicoumarol and warfarin Dicoumarol anticoagulant prevents the formation of blood clots leads to the generation of an abnormal prothrombin molecule that fails to bind calcium The effects of these vitamin K antagonists indicated the importance of vitamin K in blood clotting Normal prothrombin possess a ycarboxyglutamate residue while abnormal prothrombin formed subsequent to the administration of anticoagulants lacks this modified amino acidquot Prothrombin Binds Calcium Calcium binds to the ycarboxyglutamaterich domain of prothrombin The binding of calcium by prothrombin anchors the zymogen to phospholipid membranes derived from blood platelets after injuryquot This binding mechanism brings prothrombin into close proximity to two clotting proteins that catalyze its conversion into thrombinquot Hemophilia A Classic hemophilia Blood clotting defect In classic hemophilia factor VIII antihemophilicfactor of the intrinsic pathway is missing or has markedly reduced activityquot Action of antihemophilic factor Increased action when thrombin action decreased Regulation of Blood Clot Formation The process of clotting must be regulated Clot formation must occur only at the site of injury Blood clotting must be stopped quickly Stopping of clotting Activated clotting factors have a short life span Eg Blood clot stimulatory protein factors Va and VIIIa are shortlived due to degradation by protein C Protein C protease that is switched on by the action of thrombinquot Therefore thrombin has a dual function it catalyzes the formation of brin and it initiates the deactivation of the clotting cascade Clotting inhibitors Function to stop the formation of blood clots Eg tissue factor pathway inhibitor TFPI inhibits TF VIIa Va 22 Eg Antithrombin III is a plasma protein that inactivates thrombin by forming an irreversible complex with itquot Antithrombin 111 Also blocks other serine proteases in the clotting cascade namely factors XIIa XIa IXa and Xaquot Heparin improves the function of antithrombin III Heparin negatively charged polysaccharide found in mast cellsquot Heparin acts as an anticoagulant by increasing the rate of formation of irreversible complexes between antithrombin III and the serine protease clotting factorsquot Clots Must Be Dissolved Fibrin is split by plasminquot Plasmin serine protease hydrolyses peptide bonds Plasminogen inactive precursor of plasmin Proteolysis of plasminogen generates plasmin The activation of plasminogen is conducted by tissuetype plasminogen activator TPA TPA is bound to fibrin clots Lecture 109 Hemoglobin p 195213 Structure of Myoglobin The threedimensional structure of myoglobin was first determined in sperm whale quotConsists largely of ahelices linked to one another by turns to form a globular structure Deoxymyoglobin myoglobin without oxygen unoccupied sixth coordination site of iron in heme group Oxymyoglobin myoglobin with bound oxygen Heme prosthetic group cofactor that enables both myoglobin and hemoglobin to bind oxygen Heme Group Responsible for the red appearance of muscle and blood Components organic component central iron Fe atom Organic component Protoporphyrin Tetrapyrrole ring formed by the linkage of four pyrrole rings with methine Attached to the tetrapyrrole ring are four methyl groups two vinyl groups and two propionate groups Central iron atom The iron atom lies in the center of the protoporphyrin bonded to the four pyrrole nitrogen atomsquot Hemebound iron 9 Ferrous Fe2 or Ferric Fe3 0nIy the Fe2 state is capable of binding oxygenquot 23 The iron ion can form two additional bonds one on each side of the heme planequot the fifth and sixth coordination site Fifth coordination site in myoglobin9 imidazole ring of histidine proximal histidine Sixth coordination site site of oxygen binding Oxygen Binding Changes the Position of the Iron Ion The iron ion lies slightly outside the plane of the porphyrin in deoxymyoglobin hemequot about 04 angstroms The iron ion moves into the plane of the heme on oxygenationquot the binding of oxygen to the sixth coordination site of iron in oxymyoglobin Functional magnetic resonance imaging fMRI Method for examining brain function Differentiates oxyhemoglobin from deoxyhemoglobin Detects rearrangement of electrons upon oxygen binding to oxyhemoglobin IronOxygen Binding O O39 6 Superoxide I ion 2 3 Fe Fe hrmllvy kzmhldlhun Partial electron transfer from Fe2 to oxygen to generate Fe3 and superoxide anion Oxygen must be released as dioxygen and not a superoxide ion because 1 Superoxide and other species generated from it are reactive oxygen species that can be damaging to many biological materialsquot 2 quotThe release of superoxide would leave the iron ion in the ferric statequot and form a metmyoglobin that does not bind oxygen Myoglobin stabilizes the ironoxygen complex to prevent the leaving of oxygen as a superoxide ion A distal histidine residue present in oxymyoglobin hydrogen bonds with bound oxygen to stabilize the ironoxygen complex The protein component of myoglobin controls the intrinsic reactivity of hemequot Quaternary Structure of Hemoglobin Four myoglobinlike subunits 9 two identical alpha chains two identical beta chains Globin fold Common threedimensional fold observed in the alpha helices of hemoglobin and myoglobin Proximal and distal histidine residues are conserved in human hemoglobin and sperm whale myoglobin quotThus the alpha and beta chains are related to each other and to myoglobin by divergent evolutionquot 24 MyoglobinOxygen Binding Curve Figure 77 Simple binding curve Fractional saturation Y vs oxygen concentration measured in partial pressure p02 Fractional saturation Y The fraction of possible binding sites that contain bound oxygenquot Range 01 Initially Y increases as the concentration of oxygen increases Curve then levels off P50 Halfsaturation of the binding sites 50 of binding sites bound to oxygen At low value of 2 torr Conclusion Only a small concentration of oxygen is required for half of the total binding sites to be saturated with oxygen oxygen binds with high affinity to myoglobinquot The tight binding of oxygen to myoglobin prevents myoglobin from being an effective oxygen transport molecule like hemoglobin HemoglobinOxygen Binding Curve Figure 78 Sigmoidal curve P50 Halfsaturation of the binding sites 50 of binding sites bound to oxygen At higher value of 26 torr Conclusion Because a greater concentration of oxygen is required for half of the total binding sites to be saturated with oxygen oxygen binds with low affinity to hemoglobin Sigmoidal curve displays cooperative binding The binding of oxygen at one site within the hemoglobin tetramer increases the likelihood that oxygen binds at the remaining unoccupied sitesquot Conversely the unloading of oxygen at one heme facilitates the unloading of oxygen at the othersquot Cooperativity Enhances Oxygen Delivery By Hemoglobin Blood transports oxygen from an area of high oxygen concentration the lungs to an area of lower oxygen concentration the tissues Cooperative binding that occurs in hemoglobin 02 binding sites enables hemoglobin to transport more oxygen to the tissues from the lungs than myoglobin The cooperate release of oxygen favors a morecomplete unloading of oxygen in the tissuesquot 25 Responding to Exercise Because the change in oxygen concentration from rest to exercise corresponds to the steepest part of the oxygenbinding curve oxygen is e ectively delivered to tissues where it is most neededquot Hemoglobin is an effective oxygen transport molecule during exercise Quaternary Structural Changes on Oxygen Binding By Hemoglobin With the binding of oxygen to hemoglobin the a1 1 and az z dimers rotate about 15 degrees with respect to one another quotThe a1 1 and az z dimers are freer to move with respect to one another in the oxygenated statequot Tense T state quaternary structure of deoxyhemoglobin Relaxed R state quaternary structure of oxyhemoglobin State in which dimers are freer to move about State in which binding of oxygen occurs at higher affinity TtoR Transition By triggering the shift of the hemoglobin tetramer from the T state to the R state the binding of oxygen to one site increases the binding a inity of other sitesquot Figure 713 Concerted Model Figure 712 Explains binding cooperativity of hemoglobin Aka MWC model Iacques Monod Ieffries Wyman leanPierre Changeux States that hemoglobin can only exist in the T and R state and that binding of ligands simply shifts the equilibrium between these two statesquot The T state is strongly favored with the absence of oxygen while the R state is strongly favored with bound oxygen Sequential Model Figure 714 The binding of a ligand to one site in an assembly increases the binding affinity of neighboring sites without inducing a full conversion from the T into the R statequot Concerted vs Sequential Model Neither model in its pure form fully accounts for the behavior of hemoglobinquot The real behavior of hemoglobin is described by a combination of both models Conformational Changes in Hemoglobin Upon Oxygen Binding Oxygen binds to heme group 9 iron atom with ironassociated histidine residue at fifth coordination site moves toward porphyrin ring 9 the alpha helix with the iron associated histidine residue moves 9 the carboxyl terminal end of this alpha helix lies in the interface between the two alpha beta dimersquot 26 The change in position of the carboxyl terminal end of the helix favor the TtoR transitionquot quotStructural transition at the iron ion in one subunit is directly transmitted to the other subunits Oxygen Binding By Pure Hemoglobin vs Hemoglobin in Red Blood Cells Comparison made to study the mechanism that stabilizes the T state Pure hemoglobin binds oxygen much more tightly than does hemoglobin in red blood cellsquot Due to 23BPG 2 3BPG Anionic compound Lowers hemoglobin s affinity for oxygen About 2mM in both hemoglobin and red blood cells 23BPG binds to a central cavity a pocket in deoxyhemoglobin that is present only in the T state When transitioning to the R state the pocket collapses and 23BPG is released In order for the structural transition from T to R to take place the bonds between hemoglobin and 23BPG must be brokenquot Allosteric effector Stabilizes the T state Oxygen Affinity of Fetal Red Blood Cells Adult hemoglobin two alpha chains two beta chains Fetal hemoglobin two alpha chains two gamma chains The gamma chains in fetal hemoglobin are 72 identical to the beta chains of adult hemoglobin The gamma chains however lack a His 143 residue that stabilizes 23BPG binding in adult hemoglobin As a result the affinity of 23BPG for fetal hemoglobin is reducedquot Because 23BPG lowers hemoglobin s affinity for oxygen fetal red blood cells have a higher oxygen affinity than do maternal red blood cells because fetal hemoglobin does not bind 23BPG as well as maternal hemoglobin doesquot The Bohr Effect The regulation of oxygen binding by hydrogen ions and carbon dioxidequot Rapidly metabolizing tissues 9 Large hydrogen ion and carbon dioxide production 9 Hemoglobin triggered to transport oxygen Allosteric effectors Hydrogen ions Carbon dioxide Hydrogen ions and carbon dioxide bind to sites on hemoglobin that are not oxygen binding sites 27 The Effect of pH on the Oxygen Affinity of Hemoglobin The oxygen affinity of hemoglobin decreases as pH decreases from a value of 74quot Therefore hemoglobin is more like to release oxygen when it enters an area with a lower pH Eg The lungs have a pH of 74 while active muscle has a pH of 72 which is why hemoglobin releases oxygen when entering active muscle from the lungs Chemical groups that sense changes in pH 1 The alphaamino groups at the amino termini of the alpha chain pKa near pH 7 2 The side chains of histidines beta 146 and alpha 122 pKa near pH 7 Beta 146 Residue at C terminus of the B chain In deoxyhemoglobin the terminal carboxylate group of beta 146 forms a salt bridge with a lysine residue in the alpha subunit of the other alpha beta dimerquot This interaction locks the side chain of histidine beta 146 in a position from which it can participate in a salt bridge with negatively charged aspartate beta 94 in the same chain provided that the imidazole group of the histidine residue is protonatedquot The formation of these salt bridges stabilizes the T state leading to a greater tendency for oxygen to be releasedquot when carbon dioxide concentration are high High concentrations of carbon dioxide in a red blood cell lead to an increased formation of carbonic acid carbon dioxide water 9 carbonic acid Increased carbonic acid concentrations results in a lowered pH because carbonic acid 9 HCO3 and H Effects of Carbon Dioxide Stabilizes deoxyhemoglobin by reacting with the terminal amino groups to form carbamate groupsquot that are negatively charged These negatively charged carbamate groups participate in saltbridge interactions that stabilize the T state favoring the release of oxygenquot The Transport of CO From Tissues to Lungs Carbon dioxide is transported to the lungs as HC0339 HC0339 leaves red blood cells in specific transporters in exchange for chloride ions Reversed process in lungs HCO339 9 C02 Sickled Red Blood Cells Red blood cells deprived of oxygen 28 Lectures 1012 Bioenergetics p 427438 Principles of Metabolism 1 Fuels are degraded and large molecules are constructed step by step in a series of linked reactions called metabolic pathwaysquot 2 An energy currency common to all life forms adenosine triphosphate ATP links energyreleasing pathways with energyrequiring pathwaysquot The oxidation of carbon fuels and powers the formation of ATPquot 4 Although there are many metabolic pathways a limited number of types of reactions and particular intermediates are common to many pathwaysquot 5 Metabolic pathways are highly regulatedquot 9 Purpose of Free Energy in Organisms 1 Mechanical work Eg Muscle contraction cell movement 2 Active transport 3 Molecular synthesis Free Energy is Obtained From the Environment Phototrophs obtain energy from the sun Chemotrophs obtain energy from the oxidation of food Metabolism Chemical reactions involved in the conversion or usage of energy Metabolic pathways are interdependent communication is crucial Degradative pathways are almost always distinctquot Metabolic Pathways Catabolic reactions Catabolism Convert energy Fuel energy cellular energy Anabolic reactions Anabolism Use energy Useful energy simple precursors complex molecules Eg synthesis of glucose fats or DNA Amphibolic pathway Pathway that can be either anabolic or catabolic The construction of metabolic pathways requires 1 Specific individual reactions that will yield only one particular product or set of productsquot and 2 Thermodynamically favorable reactions 29 Thermodynamics of Metabolism A thermodynamically favorable reaction can drive a thermodynamically unfavorable reaction A shared chemical intermediate can couple a thermodynamically unfavorable reaction with a thermodynamically favorable one to drive the overall reaction Activated proteins with stored free energy can be used to drive thermodynamically unfavorable reactions Ionic gradients across cell membranes can be coupled to produce energetically unfavorable transport reactionsquot AG AG RT ln CD A B AG change in free energy AG standard freeenergy change standard condition implies the reactants A B C and D being present at a concentration of 10 M R gas constant T absolute temperature in Kelvins A B C D molar concentrations of the reactants AG AHsystemTASsystem G Gibbs free energy H Enthalpy T Temperature in kelvins K S Entropy Aern lt 0 9 Reaction is spontaneous exergonic reaction energy released Aern 0 9 System is in a state of equilibrium AH TAS Aern gt 0 9 Reaction is nonspontaneous endergonic reaction energy absorbed quotThe overall freeenergy change for a chemically coupled series of reactions is equal to the sum of the freeenergy changes of the individual steps Relationship Between AG and Kg1 AG 39 136 K39eq 10 AG expressed in kcal per mole Assume AG 39 was determined at a pH of 7 for all biochemical reactions Enzymes Do Not Alter Reaction Equilibrium Consider the reaction A B k1 10394 S391 kR 10396 S391 KBA kFkR 1039410396100 Enzymes increase both rate constants by the same factor 30 ATP Is the Universal Source of Free Energy in Biological Systems Free energy is used by organisms for mechanical work active transport and molecular synthesis ATP cellular energy carrier phosphorylgroup donor Active form of ATP ATP Mg2 or Mn quotATP is an energyrich molecule because its triphosphate unit contains two phosphoanhydride bonds ATP H20 r ADP Pi Liberated free energy AG 305 kImol 73 kcalmol ATP H20 AMP PP Liberated free energy AG 456 kImol 109 kcalmol 9AG depends on the ionic strength of medium and Mg2 or Mn2 9Liberated free energy is used to drive reactions eg muscle contraction The formation of ATP from ADP and Pi on the other hand is an endergonic process that requires an input of energy light or oxidizable substrates are used as an energy source for the synthesis of ATP ThisATP ADP cycle is the fundamen tal mode of energy exchange in biological systemsquot ATP Hydrolysis Drives Metabolism by Shifting the Equilibrium of Coupled Reactionsquot Consider the reaction A B AG 4 kcalmol K eq BeqAeq 10AG 13936 X 103 The net conversion of A9 B cannot take place when the molar ratio of B to A is equal to or greater than 115 x 10393quot But by coupling the original reaction to the hydrolysis of ATP AG 73 kcalmol the new overall reaction becomes A ATP water B ADP Pi AG 33 kcalmol 4 kcalmol 73 kcalmol 33 kcalmol At pH 7 Km BleqAleq X ADpleq Pileq ATpleq 10339313936 267 X 102 At equilibrium B A is given by the following equation BleqAleq K39eq ATpleq ADpleq Pileql Coupling of ATP hydrolysis has changed K eq BeqAeq by a factor of 105 The hydrolysis of n ATP molecules changes the equilibrium ratio of a coupled reaction by a factor of 1 08quotquot In most cells ATP ADP Pi 500 M391 Therefore BeqAeq 267 x 102x 500 134 x 105 31 A and B can be broadly defined A 9 activated conformation of protein B 9 unactivated conformation of protein Eg myosin A 9 concentration of molecule outside of cell B 9 concentration of molecule inside cell Eg sodiumpotassium pump Structural Basis of High Phosphoryl Transfer Potential of ATP ATP H20 ADP Pi AG 73 kcalmol Glycerol 3phosphate H20 glycerol Pi AG 22 kcalmol The AG for ATP is much more negative because ATP has a stronger tendency to transfer its terminal phosphoryl group to water than does glycerol 3phosphatequot ATP has a higher phosphorylgrouptransfer potential Factors that make ATP an efficient phosphorylgroup donor 1 Resonance stabilization ADP and particularly Pi have greater resonance stabilization than does ATPquot 2 Electrostatic repulsion The hydrolysis of ATP results in a reduction of repulsion between the four negative charges in ATP 3 Stabilization due to hydration More water can bind more effectively to ADP and Pi than can bind to the phosphoanhydride part of ATP stabilizing the ADP and Pi by hydrationquot The phosphoanydride bonds of ATP are high in energy and therefore release a significant amount of free energy upon hydrolysis PhosphorylTransfer Potential Compounds with a higher phosphoryltransfer potential than ATP phosphoenolpyruvate creatine phosphate 13BPG These compounds can be used in the phosphorylation of ADP for the synthesis of ATP Creatine Kinase Eg Creatine phosphate ADP ATP creatine The phosphoryltransfer potential of ATP is intermediate to the compounds mentioned above and this intermediate position enables ATP to function efficiently as a carrier of phosphoryl groupsquot ATP is analogous to currency an intermediate that does not uctuate and thus easy to store Creatine Phosphate High energy phosphoryl reservoir Creatine Kinase Creatine phosphate ADP ATP creatine AG for creatine phosphate hydrolysis 103 kcalmol AG for ATP hydrolysis 73 kcalmol AG for the formation of ATP from creatine phosphate 30 kcalmol Keq ATP creatine ADP creatine phosphate 103136 162 32 Sources of ATP During Exercise Figure 157 At the start of exercise a significant amount of ATP is consumed ATP is immediately regenerated by creatine phosphate in the initial seconds of exercise The energy released upon the hydrolysis of creatine phosphate is used for the synthesis of ATP ATP synthesis9 endergonic reaction Upon depletion of creatine phosphate ATP is generated via aerobic and anaerobic metabolism The Oxidation of Carbon Fuels ATP quotImmediate donor of free energyquot Consumed within a minute of formation The turnover of ATP is very high Resting human9 40 kg of ATP consumed in one day Exercising human9 up to 05 kg ATPmin required ATPADP cycle Motion active transport signal amplification and biosynthesis can take place only if ATP is continually regenerated from ADPquot The carbon in fuel molecules such as glucose and fats is oxidized to carbon dioxide The resulting electrons are captured and used to regenerate ATP from ADP and Piquot The AG of oxidation for methane with carbon in its most reduced form is 196 kcalmol The AG of oxidation for carbon dioxide with carbon in its most oxidized form is O kcalmol More reduced9 more free energy released The more reduced state of carbon in fats compared to those of glucose makes fats a better fuel source for ATP generation The energy of oxidation is initially trapped as a highphosphoryItransferpotentiaI compound and then used to form ATPquot Ion Gradients Stages ADP and P19 Oxidation of fuel molecules or photosynthesis 9 ATP generated 9 ATP powers the formation of ion gradients across membranes 9 ion gradients couple thermodynamically unfavorable reactions with favorable ones 9 gradient drives ATP synthesis of Extraction of Energy From Fuels Digestion Break down of food hydrolysis Proteins9 amino acids Polysaccharides 9 simple sugars Fats 9 glycerol and fatty acids 33 Degradation Conversion of smaller product obtained from previous step into even simpler units that are directly involved in metabolism Sugars fatty acids glycerol amino acids 9 acetyl CoA Small amount of ATP produced ATP production Oxidation of acetyl CoA Citric acid cycle and oxidative phosphorylation final common pathways in the oxidation of fuel moleculesquot Proton gradient is established to synthesize ATP Lecture 1014 Signal Transduction I p 401411 SignalTransduction Pathways SHIVquot Figure 141 Involves the binding of a signal molecule to a specific receptor Epinephrine betaadrenergic receptor 9 energystore mobilization Insulin insulin receptor 9 increased glucose uptake Epidermal growth factor EGF EGF receptor 9 expression of growthpromoting genes Principles of Signal Transduction 1 Signal Stimulus 9 Release of primary messenger or signal molecule Reception Primary messenger acts as a ligand and binds to extracellular region of transmembrane protein Receptor transfers information received from the signal molecule into the intracellular region of cell Delivery of the Message Inside the Cell by the Second Messengerquot Second messengers are intracellular molecules that change in concentration in response to environmental signals and mediate the next step in the molecular information circuitquot Eg of second messengers cAMP cGMP calcium ion 1P3 DAG Second messengers amplify the intracellular signal Second messengers are often free to diffuse to other cellular compartments where they can in uence processes throughout the cellquot The usage of a common second messenger in different signaling pathways can result in cross talk or input from several signaling pathwaysquot Cross talk could benefit the cell by enabling more efficient regulation of cell activity but it could also harm the cell by resulting in misinterpretation of changes in secondmessenger concentrationquot Second messengers activate many protein kinases Activation of Effectors Activation of pumps channels enzymes and transcription factors 34 5 Signaling Process Terminated Once the effectors involved in metabolic pathways are activated the signal must be terminated to enable the cell to appropriately respond to new signals Epinephrine Hormone Secreted by adrenal glands Secreted in response to internal and external stressors Epinephrine Signaling Pathway 1 Binding of epinephrine to the betaadrenergic receptor BAR Betaadrenergic receptor BAR Seventransmembranehelix 7TM receptor Receptor composed of seven helices spanning membrane Biological functions of 7TM receptors include hormone action hormone secretion neurotransmission chemotaxis etc Rhodopsin First 7TM receptor structure determined Present in retina of eye Detects photons Responsible for visual sensation Upon exposure to light rhodopsin experiences a structural change that is responsible for initiating the receptors biological function Structurally similar to threedimensional structure of Bzadrenergic receptor 2 Activation of BAR receptor Upon binding of epinephrine to the 7TM receptor BAR undergoes a conformational change 3 Activation of G protein The conformational change of BAR leads to the activation of the G protein located in the intracellular region of the cell G protein binds guanyl nucleotides G protein in unactivated state G protein is bound to GDP Heterotrimeric G protein a 8 and y subunits a subunit binds nucleotide GDP a and y subunits are bound to the membrane via attached fatty acids G protein in activated state GTP replaces GDP on a subunit upon the binding of epinephrine to BAR a subunit undergoes a conformational change upon guanine nucleotide exchange GDP 9 GTP a subunit dissociates from By dimer By dimer forms a stable complex with no enzymatic activity This dissociation of the a subunit transmits the signal that the receptor BAR has 35 bound its ligand epinephrinequot The alpha subunit of the G protein no longer has high a high affinity for G y but rather has a high binding affinity for adenylate cylase 4 Activation of adenylate cyclase Adenylate cyclase is activated by the activated G protein After GTP newly formed a subunit dissociates from the By dimer it binds to adenylate cyclase the interaction of this new alpha subunit with adenylate cyclase favors a more catalytically active conformation of the enzyme thus stimulating cAMP productionquot Adenylate cyclase Enzyme Converts ATP 9 cAMP Membrane protein with 12 membranespanning helices and two large intracellular domains that contain the catalytic apparatusquot 5 Increased cAMP 6 Activation of protein kinase A PKA and other effectors cAMP activates PKA Functions of activated PKA 1 Glycogen metabolism PKA phosphorylates two enzymes that lead to the breakdown of glycogen and the inhibition of further glycogen synthesisquot 2 Gene expression PKA alters gene expression by phosphorylating the cAMP response element binding CREE protein 3 Learning and memory PKA phosphorylation of key proteins in the synapse alter neuronal excitability and ultimately cause learning and memory at the single cell levelquot G Proteins Continuously Cycle Through Active and Inactive States The alpha subunit of the G protein has intrinsic GTPase activity This intrinsic property is responsible for the hydrolysis of bound GTP 9 GDP and Pi On hydrolysis of the bound GTP by the intrinsic GTPase activity of the alpha subunit the alpha subunit reassociates with the beta gamma dimer to form the originial heterotrimeric G protein thereby terminating the activation of adenlyate cyclasequot Signal Termination 1 Signal molecule eg epinephrine dissociates from receptor BAR 2 The phosphorylation of a specific residue in the Cterminus of the receptor leads to the deactivation of the receptor in addition quotBarrestin binds to the phosphorylated receptor and further diminishes its ability to activate G proteins 36 Phosphoinositide Cascade Also involves a 7TM receptor Eg of phosphoinositide cascade with angiotensin II receptor G protein activated in this cascade is called Gaq Gaq in GTP form binds to and activates the beta isoform of the enzyme phospholipase Cquot Second messengers diacylglycerol DAG and 145trisphosphate 1P3 are cleaved from phosphatidylinositol 45bisphosphate PIPz by phospholipase C 1P3 Shortlived messenger few seconds Dephosphorylation to inositol occurs by action of a series of phosphatases or a kinase can produce 1P2 which is then degraded to inositol by other enzymesquot Lithium is an effective inhibitor of the phosphatases resulting in stabilization of the 1P3 pool used to treat bipolar affective disorderquot 1P3 binds to specific IP3gated calcium channel proteins on intracellular membranes of endoplasmic or sarcoplasmic reticulum The binding of 1P3 to those channels results in the in ux of calcium ion into the cytoplasm from the ER 1P3 increases Ca2 Calcium ions subsequently act as a signaling molecule DAG Stays in plasma membrane Activates protein kinase C PKC DAG can only activate PKC by binding to it when PKC has bound calcium ions Calcium ions facilitate the DAGmediated activation of protein kinase Cquot Calcium Ions Properties that make calcium ions a common intracellular messenger 1 Fluctuations in the concentration of calcium ions is easy to detect Extracellular calcium ion concentration 9 SmM Cytoplasmic calcium ion concentration 9 100 nM The significantly lower intracellular concentration of calcium ions enables the easy detection of increased calcium ion concentrations within the cell 2 Calcium ion can bind tightly to proteins and induce substantial structural rearrangementsquot The capacity of Ca2 to be coordinated to multiple ligands from six to eight oxygen atoms enables it to crosslink di ferent segments of a protein and induce significant conformational changesquot Calcium Imaging Dyes such as Fura2 are used to detect intracellular changes in calcium ion concentration Because calcium ions bind well to negatively charged and uncharged oxygen atoms fura2 binds calcium ions through appropriately positioned oxygen atoms within its structurequot Fura2 bound to calcium ions will uoresce and enable detection of calcium ion 37 concentration and concentration gradients Calmodulin 17kd protein Possesses four calcium ion binding sites At cytoplasmic concentrations above about 500 nM Ca2 binds to and activates calmodulinquot EFhand protein Formed by a helixloophelix unit an EF hand is a binding site for Ca2 in many calciumsensing proteinsquot When calcium ions bind to calmodulin the protein experiences a structural change in its EF hands exposing hydrophobic surfaces that can be used to bind other proteinsquot Calcium ions act as second messengers and calmodulin acts as a secondmessenger binding protein Upon binding of calcium to calmodulin enzymes pumps and other proteins are stimulated Calcium ioncalmodulin complexes stimulate calmodulindependent protein kinases that phosphorylate many different proteins and regulate fuel metabolism ionic permeability neurotransmitter synthesis and neurotransmitter releasequot Lecture 1016 Signal Transduction II p 411422 Insulin Signaling Increased blood glucose 9 insulin insulin receptor 9 increased glucose uptake Insulin Peptide hormone Two chains linked by three disulfide bonds Insulin receptor Aka receptor tyrosine kinase Dimer Two identical units one disulfide bond One unit a chain 8 chain a subunit extracellular 8 subunit intracellular The two 1 subunits move together to form a binding site for a single insulin moleculequot The closing up of an oligomeric receptor or the oligomerization of monomeric receptors around a bound ligand is a strategy used by many receptors to initiate a signal particularly by those containing a protein kinasequot The 8 subunit contains a protein kinase domain 38 Protein kinase domain of insulin receptor The 8 subunit contains a protein kinase domain Tyrosine kinase Unlike PKA this insulinreceptor kinase catalyzes the transfer of a phosphoryl group from ATP to the hydroxyl group of tyrosine rather than serine or threoninequot Inactive without covalent modification of protein kinase domain Insulin Signaling Pathway 1 One insulin molecule binds to the insulinreceptor dimer 2 Activation of Insulin Receptor Without covalent modification of the protein kinase domain the insulin receptor is inactive An activation loop is present at the center of the protein kinase domain of the insulin receptor When the two alpha subunits of the insulin receptor come together to form an insulinbinding site the intracellular beta subunits with the protein kinase domains also come closer to each other With the two beta subunits forced together the kinase domains catalyze the addition of phosphoryl groups from ATP to tyrosine residues in the activation loops Inactive insulin receptor kinase unphosphorylated activation loops Active insulin receptor kinase phosphorylated tyrosine residues in activation loop When the tyrosine residues in the activation loop are phosphorylated the protein kinase domain experiences a structural change The kinase structure adopts a more compact conformation and this conformation is catalytically activequot J 3 Phosphorylated IRS proteins In addition to the tyrosine residues in the activation loop other sites within the receptors are also phosphorylated crossphosphorylation The other phosphorylated sites on the beta subunit of the insulin receptor are then bound by other substrates including insulinreceptor substrates IRS Eg IRS1 and IRS2 Homologous proteins Nterminus pleckstrin homology domain binds phosphoinositide phosphotyrosinebinding domain The two domains mentioned above anchor the IRS protein to the insulin receptor and the associated membranequot Both have four TyrXXMet sequences These sequences are also substrates for the activated insulinreceptor kinasequot With the phosphorylation of the tyrosine residues of the abovementioned sequences of the IRS proteins to form phosphotyrosine residues IRS molecules can act as adaptor proteinsquot 39 4 Localized phosphoinositide 3kinase The phosphotyrosine residues of the IRS proteins are recognized by Src homology 2 SHZ domains The negatively charged phosphotyrosine residue interacts with two arginine residues that are conserved in essentially all SH2 domainsquot Phosphoinositide 3kinases PI3Ks Contain SH2 domains specific to phosphotyrosine residues Bind to phosphorylated sites on IRS Add a phosphoryl group to the 3position of inositol in phosphatidylinositol 45 bisphosphate PIP2quot 5 Phosphotidylinositol345trisphosphate PIP3 Formation Phosphoinositide 3kinases bind through their SH2 domains to IRS proteins and are drawn to the membrane where they can phosphorylate PIPz to form phosphotidylinositol345trisphosphate PIP3quot 6 Activated PIP3dependent protein kinase PIP3 activates PDKl PIP3dependent protein kinase 7 Activated Akt protein kinase The activated PDKl phosphorylates and activates Akt another protein kinasequot 8 Increased glucose transporter on cell surface Akt moves through the cell to phosphorylate targets that include components that control the trafficking of the glucose receptor GLUT4 to the cell surface as well as enzymes that stimulate glycogen synthesisquot Termination of Insulin Signaling Protein tyrosine phosphatases 9 remove phosphoryl groups from tyrosine residues on the insulin receptor and the IRS adaptor proteinsquot Lipid phosphatases 9 hydrolyze PIP3 to PIPZquot Protein serine phosphatases 9 remove phosphoryl groups from activated protein kinases such as Aktquot Epidermal growth factor EGF 6 kg polypeptide Has 3 intrachain disulfide bonds Stimulates the growth of epidermal and epithelial cellsquot EGF receptor EGFR Dimer Two identical subunits Each subunit contains an intracellular protein tyrosine kinase domain that participates in crossphosphorylation reactionsquot The EGFR consists of monomers of each subunit until the binding of EGF dimerizes 40 the EGFR Each EGFR binds a single molecule of EGF in its extracellular domainquot Therefore the dimerization of the EGFR enables the binding of two EGF molecules Each EGF molecule lies far away from the dimer interface that includes a socalled dimerization arm from each monomer that reaches out and inserts into a binding pocket on the other monomerquot In an unactivated EGFR the dimerization arm binds to a domain within the same monomer that holds the receptor in a closed configurationquot Her2 receptor Structural conformation without ligand extended the ligandbound EGFR has an extended structure Overexpressed in some cancers EGF signaling pathway 1 EGF EGFR 2 Phosphorylated receptor Upon EGF binding the EGFR undergoes dimerization and the Cterminal tail of one subunit moves closer to the active site of the other subunit Cterminal tails are rich in tyrosine The Cterminal tails of the kinase domain of EGFR are phosphorylated upon EGFR dimerization Phosphotyrosines formed on EGFR 3 Binding of Grb2 Grb2 proteins contain SHZ domains that have high affinity for phosphotyrosines Grb2 also contains two SH3 domains The SHZ domains of Grb2 bind to the phosphotyrosine residues of EFGR 4 Binding of Sos Sos protein binds to SH3 domains of Grb2 5 Activated Ras Ras binds to Sos Ras Small G protein Unactivated form contains GDP Upon binding to Sos GTP replaces GDP and activates Ras Sos functions as a guaninenucleotideexchange factor GEFD 6 Activated Raf Active Ras with GTP binds Raf Raf experiences a structural change and is subsequently activated Both Ras and Raf are anchored to the membrane through covalently bound 41 isoprene lipidsquot 7 Activated MEK Activated Raf phosphorylates MEKs proteins kinases 8 Activaked ERK Activated MEK activates extracellular signalregulated kinases ERKs 9 ERK phosphorylates transcription factors 10 Changes occur in gene expression Small G Proteins Small GTPases Subfamilies Ras Rho Arf Rab and Ran Function growth differentiation cell motility cytokinesis transport of materials throughout the cellquot Cycle between an active GTPbound form and an inactive GDPbound formquot Monomeric Termination of EGF Signaling Protein phosphatases Remove phosphoryl groups from tyrosine residues on the EGF receptor and from serine threonine and tyrosine residues in the protein kinases that participate in the signaling cascadequot Ras is inactivated by its intrinsic GTPases activity GTPaseactivating proteins GAPs can speed up the conversion of activated Ras with GTP to inactive Ras with GDP GAPs facilitate GTP hydrolysis Recurring Themes of SignalTransduction Pathways Protein kinases are central to many signaltransduction pathwaysquot Epinephrine pathway cAMP dependent protein kinase Insulin and EGF pathways 9 receptors contain protein kinase domains Second messengers participate in many signaltransduction pathwaysquot Common second messengers 9 cAMP calcium ion 1P3 lipid DAG quotSpecialized domains that mediate specific interactions are present in many signaling proteins Eg Pleckstrin homology domains SH2 domains SH3 domains Signaltransduction pathways have evolved in large part by the incorporation of DNA fragmen ts encoding these domains in to genes encoding pathway componentsquot Defects in SignalTransduction Pathway Rous sarcoma virus Retrovirus 42 Causes cancer of tissues in chickens Carries vsrc oncogene that can lead to cancer csrc is a protooncogene that can become an oncogene when mutated vsrc encodes vSrc a protein tyrosine kinase that includes SHZ and SH3 domainsquot vSrc vs cSrc cSrc contains a key tyrosine residue near its Cterminal end that when phosphorylated is bound intramolecularly by the upstream SH2 domain m interaction maintains the kinase domain in an inactive conformationquot vSrc lacks the key tyrosine residue and is therefore always active Tumors often result from a mutation in a gene encoding Ras While Ras proteins must convert back to their inactive GDP form some mutations prevent this and trap Ras in its activated form and stimulate cell growth both with and without the presence of a signal molecule Tumorsuppressor genes Contribute to cancer development only when both copies of the gene normally present in a cell are deleted or otherwise damagedquot Eg genes encoding EGFsignaling pathway termination phosphatases Therapeutic Approaches Monoclonal antibodies For tumors with overexpressed receptor tyrosine kinases a therapeutic approach is to produce monoclonal antibodies to the extracellular domains of the offending receptorsquot Eg Cetuximab Erbitux is a monoclonal antibody that inhibits the overexpression of EGFR in colorectal cancers by competing with EGF for the binding site on the receptorquot Eg Trastuzumab Herceptin inhibits the overexpression of Her2 observed in breast cancers Protein kinase inhibitors as anticancer drugs Eg Gleevec inhibits BcrAbl kinase that is overexpressed in leukemia cells In chronic myelogenous leukemia parts of chromosomes 9 and 22 are reciprocally exchanged causing the bcr and abl genes to fusequot and become the bcrabl gene that overexpresses the BcrAbl kinase and causes cancer Cholera and Whooping Cough Are Due to Altered GProtein Activityquot Cholera Diarrheal disease Choleragen9 cholera toxin Result of active G protein trapped in its active state and subsequent continuous activation of protein kinase A PKA phosphorylates a chloride channel leading to its opening and phosphorylates a sodium ionhydrogen ion exchanger leading to the inhibition of sodium absorption Therefore PKA causes an excessive loss of NaCl and water 43 Pertussis Whooping cough Result of a G protein trapped in its inactive state Note Quotations indicate text obtained directly from textbook References Berg Jeremy John Tymoczko and Lubert Stryer Biochemistry 7th ed WH Freeman 2012 1 246 Print 44
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