Outline for BIOC 460 at UA
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Date Created: 02/06/15
BIOC 460 Spring 2008 Lectures 1617 Enzymes Regulation 23 Reversible covalent modification Association with regulatory proteins Irreversible covalent modificationproteolytic cleavage Readlrlg BergTymoczxo amp Stryer 6th ed ChapteriO pp 283299 Chapter is pp 3893M Problems pp 300302 Chapter 10 7 10 12 13 Key Concepts Activities of many key enzymes are regulated in cells based on metabolic needsconditions in ViiU Regulation of enzyme activity can increase or decrease substrate binding affinity andlor km 5 ways to regulate protein activity including enzyme activity i allosteric control 2 multiple forms of enzymes isozymes 3 reversible covalent modification as example phosphorylationdephosphorylation phosphorylation phosphoryl transferfrom ATP to specific 70H groups on protein catalyzed by protein kinases dephosphorylation hydrolytic removal of the phosphate groups catalyzed by protein phosphatases 4 interaction with regulatory proteins 7 examples protein xinase A PKA Caz ecalmodullnedependent xinases 5 irreversible covalent modification including proteolytic activation zymogen activation examples 7 digestive proteases lixe chymotrypsin andtrypsin 7 blood clotting cascade Learning Objectives Terminology cAlllP curiserisussequerice pseudnsubslrale cascade reclpmca reguailmi zymngen Describe in general terms how cells carry out reversible covalent modification of enzymes and how the modification would be removed Name generic names the types of enzymes that catalyze phosphorylation and dephosph orylation of proteins specify what types of amino acidfunctional groups are generally the targets of phosphorylation and show the structure of such an enzyme functional group before and after phosphorylation Explain whetherthe dephosphorylation reaction is actually the chemical reverse of the phosphorylation reaction and if not what type of reaction the dephosphorylation represents Explain the regulation of protein lltinase A PKA activity by CAMP including quaternary structural changes in PlltA triggered by CAMP binding How does the term quotpseudosubstratequot relate to the role of the regulatory subunits in PKA What are the 2enzymes involved in glycogen metabolism whose activities are reciprocally regulated by phosphorylationdephosph olylatlom Briefly discuss the structure of calmodulin Caz including structure of the EF hand motif and how Caztcalmodulin activates target proteins as an example of how a regulatory protein worllts Learning Objectives continued Describe the general mechanism by which zymogens are activated a active enzymes Briefly describe the structural change that occurs upon the activation of chymotlypslrlogerl including what changes occur in the active site Discuss the protective mechanism that llteeps prematurely activated pancreatic digestive enzymes inside the acinarcells from autodigesting the pancreas and describename an example eive examples of protease inhibitors that inhibit a elastase and b thrombin Explain how a deficiency or an inactivating chemical event in or antiproteinase formerly called ararltltrypslrl contributes to emphysema Explain how a cascade of catalysts e g y in PlltA activatlorl orin blood clotting results in amplification of a signal Outline no specificfactor namesnumbers needed how the intrinsic and extrinsic pathways converge in the final common pathway in blood clotting including roles of prothrombin thrombin fibrinogen and fibrin You don t have to learn specific namesnumbers of clotting factors in intrinsic and extrinsic pathways Name the type class of enzyme represented by most of the clotting factors Learning Objectives continued Describe the covalent modification of some of the clotting factors that requires the participation of vitamin K What is the structure of the modified amino acid R group andwhatisthe Selener abbreviation of the modified residue vvhat ion is bound by the modified amino acid resldues Briefly discuss how clots are confined to the area of lrllury including the role of heparin and antithrombin lll Describe how fibrinolysis clot breakdown is achieved with names of factors involved What is TPA7 3 Reversible Covalent Modi cation Modification of catalytic or other properties of proteins by covalent attachment of a modifying group modi cation reaction catalyzed by a specific enzyme modifying group removed by catalytic activity of a different enzyme Enzyme can cycle between active and inactive or more and less active states allosteric regulation instant sensing of local concentration signals so rapid activity changes covalent modifications generally cause slower and longer lasting effects than from allosteric regulatlorl with coordinated systemic effects e g y a hormone can trigger covalent modification events that change activities of metabolic enzymes in avariety of tissues and many cells Activities of modifyingldemodifying enzymes themselves are regulated allosterically making process sensitive to changes in concentration of small molecules that act as signals or by another reversible covalent modification process or both LEC 1617 Enzymes Regulation 2 3 BIOC 460 Spring 2008 TABLE l0l Common covalent modi cations of protein activity Example of Modi cation Donor molecule modi ed protein Protein function Phosphorylation ATP Glycogen Glucose homeostasis phosphorylase energy transduction Acetylation Acetyl CoA Histories DNA packing transcription Myristoylation Myristoyl CoA Src Signal transduction ADP ribosylation NAD RNA polymerase Transcription Farnesylation Farnesyl pyrophosphate Has Signal transduction 39yCarboxylation NCO Thrombin Blood clotting Sulfation 339Phosphoadenosine539 Fibrinogen Bloodclot formation phosphasulfate Uhiquitination Ubiquitin Cyclin Control or cell cycle Histone acetylationldeacetylation H H Histones eukaryotic proteins in chromatin KWquot CH3 involved in DNA packaging and Lquot Y regulation of gene expression very high percentages of Lys amp Arg residues lie39vlm vsiu heavily acetylated histones associated with actively transcribed genes acetylation of Lys residues catalyzed by specific acetyl transferases removal of the acetyl groups catalyzed by deacetylases modifyingdemodifying enzymes acetylases and deacetylases themselves regulated by phosphorylationdephosphorylation oc Phosphorylationldephosphorylation probably the most common means of regulating enzymes membrane channels virtually every metabolic process in eukaryotic cells Phosphomlation Kinase Any enzyme catalyzing phosphoryl transfer involving ATP or other nucleoside triphosphate named for molecule that quotreceivesquot phosphate group eg hexokinase transfers terminal phosphate from ATP to a variety of hexose sugars like glucose gt glucose6 phosphate General reaction catalyzed by kinases target ROH ATP ltgt ROPOSZ ADP Product phosphate ester of the target OH group protein kinase a generic term for kinases that transfer phosphoryl groupfrom ATP to a PROTEIN to a SerOH ThrOH or TyrOH group on the target protein Phosphorylation of enzymes is catalyzed by protein kinases Dephosphorylation phosphate group removed by hydrolysis ofphosphate ester transfer of phosphate to HZO Dephosphorylation of enzymes is catalyzed by a specific PROTEIN phosphatase Protein Kinases catalyze phosphate group transfer from ATP to a quottarget proteinquot specifically a particular OH group on the protein VERY important regulatory components in eukaryotic cells Protein kinase l 7 AG ltlt 0 equilibrium lies far to right Kinase reactions essentially irreversible maxilla Aquot can t make ATP or tyrosine this way residue 9 La l l NU on Fhosphuryloled App prololn Berg et al p 285 o 2 classes of protein kinases 1 SerineIThreonine protein kinases recipient group on target protein is a Ser OH or Thr OH 2 Tyrosine kinases recipient group on target protein is a Tyr OH Recipient target protein39s propertiesconformationactivity altered by phosphorylation Often phosphorylation causes subtle conformational change that if target is an enzyme increases or decreases catalytic activity or causes target to interact or not to interact with some other cellular component Protein kinases themselves are regulated often by allosteric effects of a small signaling molecule as in the examples below TABLE quot2 Examples of serine and threonine kinases and their activating signals Signal Enzyme Cyclic nucleotides Cyclic AMP dependent protein kinase Cyclic GMPdependent protein kinase Ca camoduin protein kinase Phosphorylase kinase or glycogen synthase kinase 2 Caquot and calmodulin AMP AMPactivated kinase Diacylglycerol Protein kinase C Metabolic Intermediates Many target specific enzymes such as pyruvate and other quotlocalquot dehydrogenase kinase and branchedihain effectors ketoacid dehydrogenase kinase PROTEIN PHOSPHATASES catalyze hydrolysis of phosphate ester bonds in phosphorylated target proteins remove the phosphate groups dephosphorylate the protein Equilibrium lies farto the right irreversible in 555 M H20 2 0 Protein 0 phosphatase V o H20 T M 039 Phosphoryluled Orthophospllute protein Pi NOTE Protein dephosphorylation is NOT the reverse of protein kinase catalyzed phosphorylation reaction Both types of reaction are irreversible Active catalyst kinase or phosphatase needed for significant reaction rates so Cell39s quotdecisionquot about what fraction of target protein is phosphorylated vs dephosphorylated depends on how active the specific protein kinase is vs how active the specific protein phosphatase is LEC 1617 Enzymes Regulation 2 3 quotCyclesquot of phosphorylationldephosphorylation hydrolyze ATP 1 Target proteinOH ATP v Target proteinOPOf ADP 2 Target proteinOPOf HZO HOPOf Target proteinOH 3 Net reaction ATP HZO ADP HOPOf hydrolysis of ATP Standard free energy change AG 39 31 lemol but Actual free energy change under cellular conditions A 39 50 lemol High negative free energy change makes Fromm 0quot ATP phosphorylationldephosphorylation cycle unidirectional in cell essential for 1 a process whose rate is being regulated Protein OPO32 ADP 2 effects of large negative AG for protein phosphorylation 1 H20 1 Some of net negative free energy Change from phosphoryl transfer makes reaction irreversible Free energy Protein OH HOP03Z 2 Some free energy is conserved in the phosphorylated protein phosphorylation of even one site on a protein can shift conformational equilibrium in protein structure by a large factor say 10 The 2 conformations can have very different catalytic or kinetic properties BIOC 460 Spring 2008 Biochemical Cascades cellularbiochemical processes with multiplicative effects Cascade a series of events in which each event in series is catalyzed by an enzyme activated in previous event First event triggered by some signal that initiates cascade eg a hormone binding to a receptor or a wound triggering the blood clotting cascade Cascade produces rapid and enormous ampli cation of original signal because every activated enzyme molecule can itself catalyze conversion of many substrates substrates often other enzymes Example Suppose one signaling molecule triggers activation of one molecule of Enzyme 1 Single molecule of active Enzyme 1 activates 100 molecules of Enzyme 2 Each of the 100 molecules of active Enzyme 2 activates 100 molecules of Enzyme 3 Each of those 10000 molecules of active Enzyme 3 activates 100 molecules of Enzyme 4 The 106 molecules of active Enzyme 4 each activates 100 molecules of Enzyme 5 we39re up to 100 million active Enzyme 5 molecules Real cascades involve a lot more than 100 products per enzyme molecule with very rapid reactions so geometric progression produces rapid and enormous response Protein Kinase Cascades Phosphorylation as a control mechanism a highly amplified effects One single activated protein kinase molecule can phosphorylate hundreds of target proteins in a very shorttime If target proteins themselves are enzymes activated by phosphorylation each activated enzyme then can carry out many many catalytic cycles on its substrate Result of cascade a major multiplicative effect between starting signal say one small molecule binds to one protein kinase molecule to activate it and final outcome several steps away Protein Kinase Specificity Some protein kinases quotmultifunctionalquot phosphorylate many different target proteins A particular kinase always phosphorylates a residue in a specific sequence or a quotconsensusquot sequence Sequences phosphorylated by that kinase very similar but not all identical example consensus sequence in all target proteins phosphorylated by protein kinase A Ser or Thr in this consensus sequence Arg Arg X Ser Z or Arg Arg X Thr Z X a small amino acid residue 2 a large hydrophobic residue Protein Kinase A binds other substrate protein sequences with a much lower affinity so doesn39t phosphorylate them very often Other protein kinases are very specific not only for local sequence but also for 3dimensional structure around it and phosphorylate only a single target protein or a small number of closely related target proteins Protein Kinase A great example of integration of allosteric regulation and regulation by reversible covalent modification phosphorylation How does cAMP activate PKA cAMP binding alters quaternary structure of protein kinase A PKA inactive form without cAMP bound 2 catalytic subunits 2 regulatory subunits regulatory subunits inhibitom CZR2 quaternary form can39t phosphorylate targets cAMP binding to R subunits makes R s dissociate from C subunits Pseudosubstrate sequence CAMp 1 39 C C i 4 CAMP i A Active Active Berg etal M Fig 1028 similarto 6th ed Fig 1017 PKA How does R binding keep C subunits inactive Specific AA sequence in R subunit of PKA that binds to the C subunit is actually a pseudosubstrate sequence Arg Arg Gly Ala lle Compare with consensus sequence where PKA phosphorylates targets Arg Arg X Ser Z or Arg Arg X Thr Z X a small amino acid residue a large hydrophobic residue But R subunit sequence has Ala instead of Ser or Thr so can39t be phosphorylated Knowing the sequence info above to what part of PKA catalytic subunits structure would regulatory subunits bind How would that binding inhibit C subunit activity Summary cAMP binds to R subunits gt conformational change affects subunit interface reduces binding affinity of R inhibitory subunits for C subunits cAM PRRcAM P complex dissociates from C subunits releasing the 2 cs Psl udmulmlldlv Individual C subunits sequulme active when free I 4 LAMP Active Aclive LAMP Structure of protein kinase A catalytic subunit bound to MgztoATP and a 20residue pseudosubstrate peptide inhibitor structure determined by Xray crvstalloqrabhv L Berg etal5th lt Fig 10 29 Berg et al tm Fig 1018 gt ATPMg part of inhibitor bound in deep cleft between 2 quotlobesquot of protein ATP bound more to one lobe inhibitor binding more to other lobe Substrate peptide binding lobes move closer together conformational changeinduced fit Restricting domain closure used to regulate protein kinase activity Essentially all known protein kinases have conserved same catalytic core residues 40280 out of 350 residues total of PKA catalytic subunit LEC 1617 Enzymes Regulation 2 3 BIOC 460 Spring 2008 Activation of Protein Kinase A by binding cyclic AMP cAMP The adenvlate cvclase cascade Example of a phosphorylation cascade in regulation Epinephrinetmusdemr 1 Regulatory cascade starts with hormone binding to extracellular 91ult39 nliVEVl 77M Newbie receptor r conformational changes in membrane proteins 39 9P 39 Ydase 2 Signal transduction communication from one protein to another gt activation of adenylate cyclase 3 adenylate cyclase enzyme catalyzing intracellular production of cyclic AMP cAMP by cyclization starting with ATP as substrate cAMP a small molecule a nucleotide 4 cAMP activates protein kinase A PKA aka quotcAMPdependent protein kinasequot cAMP an important intracellular signaling molecule in both prokaryotic and eukaryotic cells cAMP a quotsecond messengerquot signaling molecule whose production is under the control of other quotmessengersquot such as hormones coming to the cell from the extracellular environment There are other 2nd messengers in cells too with different stimuli Protein A rarein that trigger their production Rims quot55 Most effects of cAMP in cells are mediated by this one effect activation of protein kinase A Phospharylase mPhnsqhorylase 5 Active PKA then phosphoglates speci c target proteins gt many kinase km different effects in the cell Major amplification effect of PKA activation each activated molecule Berg star Fig 2115 Phosphorylase Phosplmylnu of PKA can phosphorylate a LOT of molecules of target proteins 17 a Reciprocal regulation by PKA of glycogen degradation 4 Interaction with regulatory proteins Chapter 14 pp 389391 and glycogen synthesis Example 1 Protein Kinase A PKA inactivated by binding R subunits PKA phosphorylates regulates participants in cascade leading to Example 25 C3239Cam dl1in phosphorylation of the 2 important regulated enzymes in glycogen Ca2 a ubiquitous cytosolic messenger signaling molecule metabOI39Sm Ca2 concentration quotsensedquot by Caztbinding proteins that glycogen phosphorylase breaks down glycogen when you need to get communicate signal to other proteins by proteinprotein interactions glucose out of quotstoragequot Examples glycogen synthase synthesizes glycogen when you have excess calmOdu39in caM glucose and need to store it away Troponin C TnC protein homologous to CaM in muscle cells Obviously cell doesn39t quotwantquot these 2 quotopposingquot enzymes to be active reQU39at39ng contraCtlon In response to 332 undersame conditions so they39re regulated reciprocally Calmodulin CaM M 17000 H20 Both enzymes are phosphorylated when cell wants to get glucose out example of a CaMsensing protein 0 4 Asp of storage though there39s yet another kinase activated by PKA that changes conformation when it binds ASP I actually carries out phosphorylation of glycogen phosphorylase 332 a Glycogen phosphorylase phosphorylated form more active form In Cahbound form CaM binds to and 7 Glycogen synthase phosphorylated form less active form regulates activities of many CaM 39 Result of reciprocal regulation they aren39t both Working in the cell at the dependent proteins enzymes pumps Ca Glu same time at cross purposes etc Main Reciprocal regulation of quotopposingquot pathways occurs over and over in Mode of binding of Ca2 to eamoduin chain metabolism CaM Berg et al Fig 14 13 gt I Other targets of PKA include a transcriptional activator cAMPresponse Ca coordinated to 6 O atoms from ASP 09 element binding protein CREB protein stimulates transcription of spec genes protein and 1 O atom from H 0 top CaM structure Conformational changes in calmodulin on calcium binding 4 highaffinity Ca2 binding sites ln absence of Ca1 EF hands have hydrophobic cores buried inside the each site in an quotEF handquot structural motif PFOtein EF hand motif formed by Repeating Ca binding motifs in Binding of Caz to each EF hand gt structural changes that expose helixloop helix unit structure of CaM hydrophobic patches on CaM surface common Ca2 binding CaM 2 domains each with 2 EF hand motif Cakbinding motifs Hydrophobicpatches serve as docking regions for binding target proteins Ca2 green sphere 2 domains connected by Central helix in CaM fleXible I heliX flexible even in the Ca2bound Ca state I target folds back on itself when the 2 peptide Ca domains of CaM bind to target proteins blocks access of ATP to active site in this Target proteins all have a positively charged amphipathic ahelix CaztCaM binds to positively charged amphipathic a helices in the enzymes it regulates CaM kinase peptide purple I V Target protein N 77 C CaM kinase l activated by Berg et ai Fig 445 Berg et ai Fig 3725 Berg et al Fig 141b39a CaZCAM LEC 1617 Enzymes Regulation 2 3 BIOC 460 Spring 2008 Ca2CaM binding to target enzyme s amphipathic helix stabilizes activated conformation of target enzyme After Ca2 binding step 1 2 halves of Ca2CaM clamp down around target amphipathic helix in CaM Kinase I step 2 binding it through hydrophobic and ionic interactions 5 Regulation of Enzyme Activity by Specific Proteolytic Cleavage Some enzymes biosynthesized as catalytically inactive precursor polypeptide chains Result quotextractionquot of C terminal ol helix in CaM kinase I so it s no longer Precursors fold in 3 dimensions blocking active site gt active conformation of CaM kinase l Later activated by enzymecatalyzed cleavage hydrolysis OH or conformation that can bind ATP more specific peptide bonds ZYMOGENS or proenzymes inactive precursors zymogen activation cleavageactivation process quot zquot Examples 1 mammalian digestive enzymes FaM TABLE l03 Gastric and pancreatic zymogens kinase peptide 7 GED Site of synthesis Zymogen Active enzyme Stomach Pepsinogen Pepsin Pancreas Chymotrypsinogen Chymotrypsin Pancreas Trypsinogen Trypsin Pancreas Procarboxypeptidase Carboxypeptidase Pancreas Proelastase Elastase Cnlmndulin lap Berg et al Fig 14 16b 1 digestive enzyme activation More examples of enzymesproteins activated by specific Chymotrypsin as example proteolysis Zymogen chymotrypslnogen Berg et al Figs 1020 and 1021 2 blood clotting a cascade of proteolytic activations gt rapid response Ribosomes attached to chymonypsinogen with lots of amplification endoplasmicreticulum inactive 3 some protein hormones synthesized as inactive precursors Gals 1 245 eg insulin synthesized as proinsulin complex final hormone generated by specific proteolysis to remove a peptide 4 conagen whymolrypsin T39ypSin 39 39 39 active a flbgous pgoteln wat r lnsolublet I bl Secretion 0g 7 I 1st clip activity s n eslze as roco a en a wa erso u e recursor mo ens z y p g i p lzzlianclgeatic y yrmgla i 1 15 i 15 245 5 apoptosls programmed cell death mediated by caspases acinar cells 7 7 proteases synthesized as procaspases activated by regulatory signals 6 many developmental processes controlled by precisely timed activation w Chymotrypsin f orchymotrypsin TWO diPEPtides o proenzymes active dIffuse away 1 13 16 146i 149 245 A chain B chain C chain Proteolytlc actlvatlon of chymotrypslnogen What would happen if some digestive zymogen First cleavage catalyzed by trypsin between Lys15 and lle l6 generates new aamino group on le16 Conformational change results new Nterminus of larger product chain lle16 residue turns inward and makes new salt link that molecules were activated inside pancreatic acinar cells Trypsin initiates activation ofaH the pancreatic zymogens stabilizes the active conformation Of chymotrypsin Enteropeptidase enzyme secreted by cells that line the duodenum C nf rmat39 nal Change small intestine activates a small amount of trypsinogen to trypsin 1 formation of substrate specificity Slte hydrophobic pocket where which coordinates control ofz mo en activation outside cells quotR1quot specificity group of substrate binds y g 39 2 quotcompletionquot of orientation of groups e16 What would happen if even a few zymogen molecules especially hyquot quotgt iquot 99quot trypsinogen were accidentally activated INSIDE the pancreatic II 16 acinar cells chymotrypsm to form oxyanion hole for tight binding of transition states in acylationdeacylation mechanism Chymotrypsinogen t E lal 1activi What prevents premature activation of pancreatic zymogens inside cells Small very specific very tightbinding inhibitor proteins inside cell T inhibit any protease molecule that39s accidentally prematurely wChymolrypsin ryps39quot activated active m Berg et 3L example pancreatic trypsin inhibitor PTI 6000 MW Fig 10 22 LEC 1617 Enzymes Regulation 2 3 BIOC 460 Spring 2008 quotPTIquot binds VERY tightly to trypsin not even 8 M urea or e M Another tightbinding protease inhibitor helps prevent guanidine HCI dissociate the complex emphysema Part of PTI binds in active site of trypsin with a Lys residue of PTI occupying R1 quotspecificity pocketquot Emphysema results from loss of elasticity elastic fibers and other connective tissue proteins in alveolar walls of the lungs so CO2 can39t be exhaled effectively so there isn39t room for inhaling much fresh air 02 Neutrophils white blood cells that engulf invading bacteria secrete elastase Excess elastase in blood plasma can hydrolyze elastic fibers in alveolar walls of the lungs emphysema To prevent elastase from running amok in plasma liver makes and secretes a plasma protein a1antiproteinase used to be called quotoz antitrypsinquot but that39s a misnomer it binds much tighter to elastase than to trypsin aantiproteinase in blood plasma keeps elastase inhibited protecting lungs from damage Pancreatic trypsin inhibitor is a substrate but the peptide bond quotafterquot that Lys is cleaved only VERY slowly time scale of months Combination of very tight binding and very slow catalytic turnover makes PTI a very effective inhibitor quot quot Trypsmpancreatl trypsm Free pancreatic 39 complex trypsin 39 INTRINSlC PATHWAV 2 BLOOD CLOTTING 3 Consequences of oqantiproteinase deficiency Damaged surface firrfisnigtofggggv Vtgszgfs fhe39ia39 cascade ofiprotedy c Inherited disorders defects either in its structure making it less Kininogequot Sinis s iyfggt lgion effective as an inhibitor or slowing down its secretion from liver and Kamkrein Triggered by SHbS aMFS g released from tissues m 0f Wh e serles 0f thus reducing its concentration in plasma W responsem uma clotting factors named 1 genetic deficiency in a1antiproteinase increased 39 EXTRINSIC PATNWAV W39th ROW numerals 39n babilit of develo in em h sema quot 9 order of dlsmvery not In pr y p 9 p y 39 r order in which they work iii a m 39in in cascade 2 Cigarette smoke damages the inhibitor I I mm Mum cascades that converge Component of cigarette smoke OXIdIZeS a Met residue in 39 quotno nal C mm n aantiproteinase that39s required for binding to elastase r Pathway oxidation nonfunctional inhibitor 339 quot product of quotfinal common Result smokers continually inactivate aantiproteinase in 1939 PathwaYquot fibrin 03901 their lungs and thus are also much more likely to develop Very rapid process huge emphysema amplification of original signal enormous response lmaginle the results of a combination of a genetic deficiency and cigarette JI39MEN Twas Genetic de ciency in any smoke PATHWAY 1 clotting factor hemophilia 39 activated cigs39nlliy iked quoty b ncln Berg etal Fig 1026 Blood clotting continued 0 Blood clotting continued Final steps in blood clotting understood at molecular level no details here f V39ca39bc xyg39u ama39e 1 Thrombincafayzed profeoysis of fibrinogen soluble fibrin c H H o monomers whlk jc39 2 Selfassociation of fibrin monomers insoluble protofibrils quotsoft quot HZ clotsquot em 3 Covalent crosslinking of fibrin protofibrils final clot o oo coo ynvbuxyglulumn39o residue Why does vitamin K deficiency lead to slow blood clotting Ca2 binding region of prothrombin Vitamin K required for activity of glutamate carboxylase make Gla m di ed R groups Gla in ball 8 StiCk Posttranslational modification of specific Glu residues in 4 of the clotting 39 o aims f carbozfy groups on Gla Jo factors gt ycarboxyglutamate addition of carboxyl group res39dues red ca 39 ns green 9 Multiple Gla residues in zymogens close in primary structure bind Vitamin K analogs 332 ions Coumarins eg dicoumarol drugs Calcium ions Ca2 com lexes bind ne char ed hos holi ids on latelet anti39domng agentsv anticoagu39amst surfacesp g g p p p p prevent heart attacks and strokes 2 Don39t inhibit carboxylase inhibit ZymogenGla Ca platelet complexes hold and orient clotting another enzyme needed to recycle factors in exact location where they need to be activated where platelets have bound to injured blood vessel wall and clot is needed Thus high concentration of active thrombin forms at wound site vitamin Kfor repeated use Warfarin analog used as a rat poison Berg e aIvFi9103932 LEC 1617 Enzymes Regulation 2 3 BIOC 460 Spring 2008 Removal of clot itself Tissuetype plasminogen activator TPA a serine protease Has a fibrin binding domain that targets it to fibrin clots where it Regulation of Blood Clotting Too little or too slow clotting gt hemorrhage potentially fatal Too much or inappropriately located clots gt heart attacks strokes thrombosis also potentially fatal finds plasminogen Requirements Plasminogen binds to fibrin clots too CIOtS haVe to form raPIdIY TPA cleaves plasminogen gt active plasmin another serine protease cm have to be local39zed at S39te 0f 39nJury Clots dissolved by plasmin which cleaves fibrin in clots Clomng faCtOFS haVe to be remOVed qUICkly after CIOt format39on Gene for TPA has been cloned used for producing TPA in cultured T f f I tr d mammalian cells erm39na 39 quot C 39ng casca IV administration of TPA within an hour of clot formation in a RemOVal Of 011an faCtOTS coronary artery heart attack markedly increases patient s chance 1 Dilution by blood flow of survival 2 Removal by liver 3 Protease degradation eg protease C activated by thrombin so final steps in clot formation also prevent spread of clotting beyond wound area 4 Binding to specific inhibitors eg antithrombin III a protease inhibitor in blood plasma binds thrombin and other serine proteases in clotting cascade tightly in presence of heparin a negatively charged polysaccharide heparin binding gt conformational change in antithrombin III that increases rate of binding to clotting factors it inhibits Arrow marksiclot position in lefthand fi ure Berg etal Fig 1036 Before TPA After TPA LEC 1617 Enzymes Regulation 2 3
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