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Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
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Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
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Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
Molbiol 2011-11-role of-proteins
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Molbiol 2011-11-role of-proteins

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  • 1. Globular Proteins
  • 2. Globular proteins are characterized as generallyhaving:•  a variety of different kinds of secondary structure•  spherical shape•  good water solubility•  a catalytic/regulatory/transport role i.e. a dynamic metabolic function
  • 3. Globular heme proteins contain heme as prostheticgroup.Functions of globular hemeproteins include: •  electron carriers •  part of enzyme active site •  transport of O2 and CO2- hemoglobin •  storage of O2-myoglobin
  • 4. •  II.  Globular  Hemeproteins  •  Contain  heme  as  prosthe.c  group  •  Role  of  heme  is  dependent  on  environment  created  by   3D  structure  of  protein  •  Heme  of  cytochrome  →  electron  carrier  •  Heme  of  catalase  →  part  of  ac.ve  site  •  Heme  of  Hb  and  myoglobin  →  binds  O2  reversibly  
  • 5. •  A.  Structure  of  Heme  •  Complex  of  Protoporphyrin   IX  &  Fe2+    •  Fe2+  bound  to  4  Ns,  other   2  bonds  perpendicular  to   plane  of  ring  available  for   bonding  •  In  Hb,  one  of  these   aHached  to  N  terminus  of   His,  other  binds  O2.    
  • 6. Structure of heme porphyrin heme (Fe-protoporphyrin IX)
  • 7. heme“proximal” histidine “distal” histidine
  • 8. B.  Structure  and  func9on  of  myoglobin    •  It  is  a  heme  protein  present  in   heart  and  skeletal  muscle  •  Reservoir  for  O2  and  carrier  of   O2  in  muscle  cell  •  Single  polypep.de  chain   similar  to  polypep.des  in  Hb  •  1.  α-­‐helical  content:  •  ~  80%  of  pep.de  in  8   stretches  of  α-­‐helix  Labeled  A   to  H  •  Terminated  by  Pro  or  β-­‐bends   and  loops  stabilized  by  H   bonds  and  ionic  bonds.  
  • 9. •  2.  Loca9on  of  polar  and  nonpolar  amino  acid  residues:  •  Interior  made  up  of  hydrophobic  amino  acids  stabilized  by   hydrophobic  interac.ons  •  Surface  →  charged  amino  acids  –  form  H  bonds  with  water    •  3.  Binding  of  heme  group:  •  Heme  in  crevice  lined  with  non-­‐polar  amino  acids,  except  2   His  residues  •  Proximal  his9dine  –  binds  directly  to  Fe2+  of  heme  •  Distal  his9dine  stabilizes  binding  of  O2  to  Fe2+  
  • 10. O2 Binding in Mb andHb
  • 11.   C.  Structure  and  func9on  of  hemoglobin    •  Found  exclusively  in  RBCs  →   transports  O2  •  Hb  A  –  predominant  form  in   adults:  4  polypep.de  chains   -­‐-­‐  α2β2  •  Each  subunit  –  heme-­‐binding   pocket  similar  to  myoglobin  •  Can  transport  O2  and  CO2  •  O2-­‐binding  proper.es   affected  by  allosteric   effectors,  unlike  myoglobin  
  • 12. 1.  Quaternary  structure  of  hemoglobin:     •  2  iden.cal  dimers:  (αβ)1  and   (αβ)2   •  dimers  held  together  by   hydrophobic  interac.ons  (on   contact  surfaces  of  subunits   as  well  as  internally)  but   ionic  and  H-­‐bonding  also   exist     •  2  dimers  held  together  by   weak  polar  bonds     •  different  conforma.on  in   deoxyHb  and  oxyHb  
  • 13. αβ dimer 2 αβ dimer1
  • 14. T and R forms of Hemoglobin T = “taut” → deoxy Hb → low affinity for O2 R = “relaxed” → oxy Hb → high affinity for O2
  • 15. •  a.  T  form:  “taut”  form  •  deoxy  form  of  Hb  •  2  αβ  dimers  joined  by  ionic  and  H-­‐bonds  •  low  oxygen-­‐affinity  form  of  Hb  •  b.  R  form:    •  binding  of  O2  disrupts  some  ionic  and  H-­‐ bonds  between  αβ  dimers    •  “relaxed”  form  •  high  oxygen-­‐affinity  form  of  Hb  
  • 16.   D.  Binding  of  oxygen  to  myoglobin  and   hemoglobin    •  D.  Binding  of  oxygen  to  myoglobin   and  hemoglobin  •  Myoglobin  →  one  heme  →  binds   one  O2  •  Hb  →  4  heme→  binds  4  O2  •  Hb  binding:  degree  of  satura.on   (Y)  from  0  to  100%  •  1.  Oxygen  dissocia9on  curve:  •  plot  of  Y  against  PO2  •  myoglobin  :  higher  affinity  for  O2   than  Hb  •  P50  is  1  mm  Hg  for  myoglobin  and   26  mm  Hg  for  Hb  
  • 17. •  a.  Myoglobin:  •  O2  dissocia.on  curve  hyperbolic  •  This  reflects  that  myo  binds  single  O2  •  Mb  +  O2              MbO2  they  exist  in  equilibrium  •  Exchange  between  Hb  and  Mb,  Mb  and   muscle  cells  depending  on  equilibrium  •  Mb  binds  O2  released  from  Hb,  releases   when  O2  drops.    Mb  then  releases  the  O2   into  the  muscle  cell.    This  only  happens  when   there  is  an  O2  demand.  
  • 18. •  b.  Hemoglobin:  •  O2  dissocia.on  curve  is   sigmoidal    •  Coopera.ve  bind  of  O2   (increased  affinity  for  Hb   with  more  binding)  •  Heme-­‐heme  interac.on:   binding  of  O2  at  one  heme   increases  affinity  for  O2  at   others  
  • 19. •  E.  Allosteric  effects  •  Ability  of  Hb  to  bind  O2  depends  on  allosteric   (“other  site”)  effectors:   –  PO2   –  pH  of  environment   –  PCO2-­‐  an  inc  will  cause  the  inc  in  unloading  of  O2.   –  2,3-­‐disphosphoglycerate  availability  •  allosteric  factors  do  not  affect  myoglobin  
  • 20. •  1.  Heme-­‐heme  interac9ons:  •  structural  changes  in  one  heme  group  transmiHed  to   others  •  affinity  for  last  O2  ~300X  affinity  for  first  O2  •  a.  Loading  and  unloading  of  oxygen:  •  more  O2  can  be  delivered  to  .ssues  with  small   changes  in  PO2  •  Graph  showing  loading  and  unloading  at  different   par.al  pressures  of  O2.  Hb  alterna.vely  carries  O2   and  CO2  between  lungs  and  .ssues    •  b.  Significance  of  sigmoidal  O2-­‐dissocia9on  curve   Compare  a  hyperbolic  curve  to  a  sigmoidal  curve  •  A  sigmoidal  curve  gives  increasing  affinity  of  O2  for  Hb   with  increasing  par.al  pressure  while  a  hyperbolic   curve  is  a  straight  line  in  that  range.  
  • 21. •  2.  Binding  of  CO2:  •  Most  of  the  CO2  in  the   blood  is  transported  as   bicarbonate:  •  CO2  +  H2O            H2CO3  •  H2CO3                  HCO3-­‐    +  H+  •  Some  CO2  binds  to  the   terminal  –NH2  of  the  α   and  β  chains  forming   carbaminoHb.  •   Binding  of  CO2  stabilizes   the  “taut”  form  of  Hb   (deoxyHb).    
  • 22. •  3.  Binding  of  CO:  •  CO  binds  reversibly  to  the  Fe2+  the  same  way   that  O2  does  •  CO  +  Hb    HbCO  (carbon  monoxy  Hb)  •  Affinity  of  Hb  for  CO  is  220X  affinity  for  O2  •  Binding  of  CO  to  Hb  increases  affinity  of   remaining  sites  for  O2  •  O2  dissocia.on  curve  shigs  to  leg  (becomes   hyperbolic)  •  >  60%  HbCO  fatal  •  treated  with  O2  therapy    
  • 23. 4.  Bohr  Effect:    •  Shig  of  O2  dissocia.on   curve  to  the  right  with   decrease  in  pH  or  increase   in  PCO2    •  This  translates  to  a   decreased  affinity  of  Hb   for  O2  under  these   condi.ons,  therefore  you   unload  O2  easier  
  • 24. •  a.  Source  of  the  protons  that  lower  the  pH:  •  2  principle  sources  of  protons:   –  lac.c  acid  produced  by  anaerobic  metabolism  in  muscles   –  increased  produc.on  of  CO2  by  cell  u.liza.on  of  O2  through   respira.on:  •  CO2  +  H2O              H2CO3                  H+  +  HCO3-­‐   –  in  lungs  the  equilibrium  of  this  reac.on  is  towards  the  leg   because  CO2  is  lost  through  exhaling  •  the  decreased  affinity  of  Hb  for  O2  under  the  Bohr   effect  condi.ons  results  is  greater  off  loading  (release)   of  O2  in  the  .ssues.  
  • 25. The Effect of CO2 and H+ on O2 Binding Bohr Effect: Increased concentrations of CO2 and H+ promote the release of O2 from hemoglobin in the blood.
  • 26. How do CO2 and H+ promote the release of O2from hemoglobin?•  presence of “salt bridge” •  no ionic interaction in in T form R form
  • 27. CO2 is bound to hemoglobin at protein interfaces, notFe2+ center!
  • 28. •  Summary  reac.on  for  the  Bohr  effect:  •  HbO2  +  H+              HbH+  +  O2            OxyHb                      DeoxyHb        •  Equilibrium  shigs  to    the  right  when  H+  conc.   increases  (decrease  in  pH),  while  it  shigs  to   leg  when  PO2  increases.        •  The  protonated  forms  of  the  terminal  α-­‐ subunit  –NH2  groups  and  His  side-­‐chains   stabilize  the  T  form  (deoxy  form)  of  Hb.  
  • 29. •  5.  Effect  of    2,  3-­‐bis-­‐ phosphoglycerate(BPG)  on   oxygen  affinity:  •  Important  regulator  of  Hb   binding  O2  •  Most  abundant  organic   phosphate  in  RBC  (conc.  ~  =   conc.  of  Hb)  •  Synthesized  from   intermediate  of  glycolysis    •  a.  Binding  of  2,3-­‐BPG  to   deoxyhemoglobin:  •  Binds  to  deoxyHb  stabilizing  it  •  Decreases  affinity  of  Hb  for  O2  
  • 30. •  b.  Binding  site  of  2,3-­‐BPG:  •  1  molecule  of  2,3-­‐BPG  binds  to  a   pocket  between  the  β-­‐chains  in   the  center  of  the  deoxyHb  center  •  expelled  on  oxida.on  of  Hb   (pocket  disappears)  •  c.  ShiX  of  oxygen-­‐dissocia9on   curve:  •  Blood  stripped  of  2,3-­‐BPG  has  a   high  affinity  for  O2  •  2,3-­‐BPG  shigs  the  O2-­‐dissocia.on   curve  to  the  right  allowing   decreased  affinity  of  Hb  for  O2   and  effec.ve  unloading  of  O2  in   .ssues  •  similar  to  Bohr  effect  but  no   difference  between  lungs  and   .ssues  
  • 31. •  d.  Response  of  2,3-­‐BPG  levels  to  chronic   hypoxia  or  anemia:  •  2,3-­‐BPG  increases  in  chronic  hypoxia    •  chronic  hypoxia  can  be  caused  by     –  pulmonary  emphysema  or     –  high  al.tudes  or   –  chronic  anemia    •  increased  2,3-­‐BPG  shigs  O2  dissocia.on   further  to  right  allowing  greater  unloading   of  O2  
  • 32. •  e.  Role  of  2,3-­‐BPG  in  transfused  blood:  •  2,3-­‐BPG  essen.al  for  normal  transport  func.on  of   blood  •  Without  normal  concs.  of  2,3-­‐BPG,  Hb  becomes  an   O2  trap  (doesn’t  unload;  high  affinity)  •  Blood  for  transfusion  formerly  stored  in  acid-­‐citrate-­‐ dextrose  medium  decreased  2,3-­‐BPG  conc.  →   “stripped”  blood  •  Body  restores  conc.  of  2,3-­‐BPG  in  24  –  48  h  •  2,3-­‐BPG  can  be  restored  by  adding  inosin  
  • 33. Minor Hemoglobins
  • 34. Minor Hemoglobins
  • 35. Minor HemoglobinsEmbryonic form is Hb Gower 1(ζ2ε2) (yolk sac).HbF - 2 α chains, 2 γ chains (β-chain family) - major form infetus and newborn (fetal liver –2 weeks).HbA - 2 β chains, 2 α chains -major form in adult.Fetal bone marrow beginssynthesizing HbA around 8thmonth.
  • 36. Globin gene organization
  • 37. Steps in globin chain synthesis:1.  Transcription2.  Modification of mRNA precursor by splicing3.  Translation by ribosomes & further modifications (i.e. glycosylation)
  • 38. Hemoglobinopathies•  caused by abnormal structure of Hb•  characterized by low levels of normal Hb Sickle-cell anemia (Hemoglobin S disease) Hemoglobin C disease Hemoglobin SC disease Thalassemias – α thalassemia β thalassemia
  • 39. Sickle-cell anemia (HbS disease)•  abnormal β chain. HbS = α2βS2•  β chain mutation - glu 6 à val 6•  glu is negatively charged, val is nonpolar.•  only has effect postnatally because HbF is majorspecies in fetus•  symptoms - hemolytic anemia, painful crises,poor circulation, frequent infections•  heterozygotes - HbA and HbS both present - 1 in10 African Americans; "sickle cell trait" - nosymptoms, normal life span
  • 40. Sickle-cell anemia (HbS disease)•  glutamic acid is replaced by valine at position 6 of βchain
  • 41. normal RBCs sickled RBCs
  • 42. Symptoms worsen when Hb is in deoxy form - decreased pO2,increased CO2, decreased pH, increased 2,3-BPG
  • 43. Low solubility of HbScauses aggregation anddistortion of cell shape.
  • 44. HbS•  val instead of glu atposition 6HbA•  glu at position 6HbC•  lys instead of glu atposition 6HbSC•  HbS as well as HbCpresent → 2 bands inelectrophoresis
  • 45. HbC disease•  lys instead of glu at position 6•  HbC homozygotes - mild, chronic hemolytic anemia. Not life-threatening HbSC disease •  HbS as well as HbC present → 2 bands in electrophresis •  usually undiagnosed until infarctive crisis occurs (childbirth, surgery) •  can be fatal
  • 46. Thalassemias•  hereditary hemolytic diseases•  most common genetic disorder in humans•  heterogeneous collection of diseases
  • 47. β-thalassemias•  synthesis of β-chain decreased or absent β-thalassemia minor (or trait) - one normal, one defective β- chain gene. Not life-threatening β-thalassemia major - both genes defective. Normal at birth. Severe anemia by age 1-2. Treatment requires frequent transfusions → Leads to iron overload (hemosiderosis). Death between 15-25 years old. Bone marrow transplant (BMT) is an option.
  • 48. α-thalassemias•  decreased or absent α chain synthesis•  severity of disease depends upon the numberof defective α genes:0 defective - normal1 defective - silent carrier of α-thalassemia. Nosymptoms2 defective - α-thalassemia trait - no serioussymptoms3 defective - Hemoglobin H disease - moderatelysevere hemolytic anemiaall 4 defective - hydrops fetalis - fetal death (αchains needed for HbF)
  • 49. Methemoglobinemia  •  1.  Forma9on  of  methemoglobin  •  Oxida.on  of  Fe2+  →  Fe3+  converts  Hb  and  myoglobin  to   metHb  and  metmyoglobin  •  Cannot  bind  O2,    •  Oxida.on  by  drugs  like  nitrates,  H2O2  or  free  radicals  or   muta.on  in  α-­‐  or  β-­‐chain  of  globin  →   methemoglobinopathy  (HbM).  •  a.  Reduc9on  of  methemoglobin:  •  Normal  oxida.on  corrected  by  NADH-­‐cytochrome  b5-­‐ reductase  •  RBCs  of  newborns  →  half  the  capacity  of  this  enzyme,   therefore  more  suscep.ble  to  oxida.on  
  • 50. Fibrous Proteins
  • 51. Fibrous  proteins  are  characterized  as  generally  having:    •     one  domina.ng  kind  of  secondary  structure          (i.e.  collagen  helix  in  collagen)  •     a  long  narrow  rod-­‐like  structure  •     low  water  solubility  •     a  role  in  determining  .ssue/cellular  structure  and        func.on  (e.g.  collagen,  α-kera.n)  
  • 52. Collagen  -­‐  most  abundant  protein  in  body;  rigid,  insoluble      Elas.n  -­‐  stretchy,  rubber-­‐like,  lungs,  walls  of  large  blood  vessels,  ligaments      
  • 53. Structure  of  Collagen   Tropocollagen  is  a  right-­‐handed  triple  helix     formed  of  α-­‐chains.  
  • 54. Structure  of  Collagen  The  α-­‐chains  (individual  polypep.des  composing  tropocollagen)  consist  of  -­‐[Gly-­‐X-­‐Y]-­‐    repeats.    Proline  and  hydroxyproline/hydroxylysine  are  ogen  present  in  the  X  and  Y  posi.ons,  respec.vely.  
  • 55. Synthesis  of  collagen    •     made  in  fibroblast,  osteoblasts  (bone),  chondroblasts  (car.lage)  •     secreted  into  ECM  •     enzyma.cally  modified  •     aggregate  and  are  cross-­‐linked  
  • 56. Structure  of  tropocollagen  molecule  
  • 57. Biosynthesis  of  collagen  1.  forma.on  of  pro-­‐α-­‐chains  -­‐  contains  signal  sequence  –   promotes  binding  of  polysome  to  RER  and  secre.on  into  the   cisternae;  signal  sequence  removed  2.  some  pro  and  lys  residues  (in  the  Y  posi.on  of  gly-­‐X-­‐Y)  are   hydroxylated  by  prolyl  hydroxylase  and  lysyl  hydroxylase;   needs  molecular  O2  and  reducing  agent  like  ascorbic  acid   (from  vitamin  C).  3.  glycosyla.on  -­‐  glucose  and  galactose  added  to   hydroxylysines;  pro-­‐α-­‐chains  join  to  form  procollagen.  N-­‐  and   C-­‐terminal  extensions  form  interchain  disulfide  bonds;  central   triple  helix  formed  because  of  favorable  alignment;   Transported  to  Golgi,  packaged,  and  secreted  as  procollagen.  
  • 58. Biosynthesis  of  collagen  
  • 59. Biosynthesis  of  collagen  (cont’d)   4.    N-­‐procollagen  pep.dase  and  C-­‐procollagen  pep.dase  remove   terminal  extensions,  leaving  triple  helical  collagen  (occurs   extracellularly).   5.    collagen  fibrils  -­‐  form  by  associa.on  of  collagen  molecules   with  about  a  3/4  overlap  with  other  molecules  (staggered,   parallel  arrays)   5.    cross-­‐linking  -­‐  interchain  cross-­‐links  caused  by  lysyl  oxidase  (a   pyridoxal  phosphate  and  copper-­‐requiring  enzyme);  O2   required;  oxida.ve  deamina.on  of  lysines  and   hydroxylysines;  Allysine  (aldehyde)  reacts  with  amino  group   of  nearby  lysine  or  hydroxylysine  to  form  interchain  cross-­‐ link.  Very  important  for  tensile  strength  of  collagen.  
  • 60. Ascorbate  coenzyme  required  by  prolyl/lysyl  hydroxylase  in  hydroxyla.on  step.  Vitamin  C  (ascorbate)  deficiency  results  in  scurvy  (collagen  can’t  be  cross-­‐linked).  
  • 61. Cross  links  formed  by  lysyl/ Cu2+/  prolyl  oxidase   vitamin  B6    -­‐  copper  coenzyme    Number  of  cross-­‐links  increases  with  age  →  causes  s.ffening,  decreased  elas.city  of  skin  and  joints.  
  • 62. Biosynthesis  of  collagen  (con’t)   In  the  final  step,  collagen  fibrils  form  spontaneously  from   tropocollagen.  covalent  X-­‐links  between  Allysine  and  hydroxylysine     tropocollagen   molecule   triple  helix  of   α-­‐chains.  
  • 63. Types  of  Collagen   CommonType Representative Tissues disorders Ehlers-Danlos Osteogenesis I Imperfecta skin, bone, tendons, cornea Marfan’s cartilage, intervertebral disks, vitreous II - body blood vessels, lymph nodes, dermis, III Ehlers-Danlos early phases of wound repair Alport’s IV basement membranes Goodpasture’s X - epiphyseal plates
  • 64. Collagen  Degrada.on  and  Disorders   •     degrada.on  of  collagen  by  collagenase  allows   remodeling  of  ECM   Ehlers-­‐Danlos  –  hyperextensive  joints,  hyperelas.city  of   skin,  aor.c  aneurisms,  rupture  of  colon,  skin   hemmorhages  due  to  muta.on  in  α-­‐chains     Osteogenesis  Imperfecta  –  briHle  bone  disease,  mul.ple   fractures,  blue  sclera,  hearing  loss,  retarded  wound   healing    
  • 65. Ehlers-­‐Danlos  Syndrome    Hyperextension  of  skin  
  • 66. Osteogenesis  Imperfecta     (Blue  sclera)  
  • 67. In  Utero  Radiograph:  •     crumpled  long  bones  •     beaded  ribs  
  • 68. Elas.n   •     rubber-­‐like  proper.es   •     connec.ve  .ssue  protein   •     lungs,  large  blood  vessels,  elas.c  ligaments    Composi.on:        -­‐  small  nonpolar  amino  acids  (Gly,  Ala,  Val)      -­‐   also  rich  in  Pro  and  Lys    -­‐  liHle  or  no  OH-­‐Pro  or  OH-­‐Lys    
  • 69. Elas.n  
  • 70. Elas.n  •     3D  network  of  cross-­‐linked  polypep.des  •     cross  links  involve  Lys    and  alLys    •     4  Lys  can  be  cross-­‐linked  into  desmosine    •     desmosines  account  for  elas.c  proper.es  
  • 71. Elas.n  Degrada.on  and  Disorders  •     in  lungs  -­‐  lung  alveolar  elas.n  in  constantly  exposed  to  neutrophil  elastase        α1-­‐AT  inhibits  elastase  thus  preven.ng  loss  of  lung  elas.city  •     individuals  who  are  homozygotes  for  mutant  α1-­‐AT  are  very  suscep.ble  to  emphysema  
  • 72. Enzymes
  • 73. Enzymes arebiological catalysts.
  • 74. Some nomenclature…Active site = special pocket where substrate bindsSpecificity1.  enzymes are specific for a single molecule or a structurally related group of substrates2. usually only 1 enzyme per reaction type
  • 75. Some more nomenclature…Cofactor = inorganic component needed for enzyme function
  • 76. Some more nomenclature…Coenzyme = nonprotein small organic componentneeded for enzyme function
  • 77. Some more nomenclature…Holoenzyme - the enzyme protein plus its cofactorApoenzyme - enzyme protein without its cofactorProsthetic groups – a coenzyme that’s very tightly (usually covalently) attached to the protein, such as heme
  • 78. How Enzymes Work Enzymes increase the rate of reactions without themselves being altered in the process of substrate conversion to product. This defines a catalyst.   Enzymes increase reaction rates by lowering the energy input needed to form a reactant complex that will eventually form product.   This occurs via the formation of a complex between enzyme and substrate (ES): k1 k2 E + S ES E + P k-1
  • 79. Steps in an Enzymatic Reaction1.  Enzyme and substrate combine to form a complex.2.  Complex goes through a transition state – not quite substrate or product3.  A complex of the enzyme and the product is produced.4.  Finally, the enzyme and product separate.All of these steps are equilibria.
  • 80. Steps in an Enzymatic Reaction
  • 81. Steps in an Enzymatic Reaction1.  Enzyme and substrate combine to form a complex.
  • 82. Steps in an Enzymatic Reaction2. The complex goes through a transition state – not quite substrate or product
  • 83. Steps in an Enzymatic Reaction3.  A complex of enzyme and product is produced (EP).4.  The product is released.
  • 84. Factors that influence enzyme activity Environmental factors •  temperature, pH Cofactors •  metal ions Effectors •  species that alter enzyme activity
  • 85. Effect of pH on enzyme activity
  • 86. Effect of pH on enzyme activity Examples of optimum pH
  • 87. Effect of temperature on enzyme activity •  exceeding normal temperature ranges always reduces enzyme reaction rates •  optimum temperature is usually 25 - 40 ºC (but not always)
  • 88. Kinetics•  Kinetics is the study of the rate of change ofreactants to products•  Velocity (v) refers to the change in conc. ofsubstrate or product per unit time•  Rate (k) refers to the change in total quantity (ofreactant or product) per unit time•  Initial velocity (v0) is the change in reactant orproduct conc. during the linear phase of a reaction
  • 89. Michaelis-Menten KineticsThree basic assumptions:1: ES complex is in a steady state, i.e. remains constant during the initial phase of a reaction2: when enzyme is saturated all enzyme is in the form of ES complex3: if all enzyme in ES then rate of product formation is maximal: Vmax = k2[ES]  
  • 90. Michaelis-Menten Kinetics The Michaelis-Menten equation is a quantitative description of the relationship between the rate of an enzyme catalyzed reaction (v1), substrate concentration [S], the M-M rate constant (Km) and maximal velocity (Vmax)
  • 91. Michaelis-Menten Kinetics Km is equal to the concentration of substrate required to attain half maximal velocity for any given reaction
  • 92. Lineweaver-Burk Analysis•  Lineweaver and Burk manipulated the MMequation by taking its reciprocal values generating adouble reciprocal plot•  Leads to a linear graph of the reciprocals ofvelocity and substrate concentration
  • 93. Lineweaver-Burk Plot
  • 94. Enzyme inhibition •  many substances can inhibit enzyme activity: substrate analogs toxins drugs metal complexes
  • 95. Enzyme inhibition - 2 broad classes:Irreversible inhibition•  forms covalent or very strong noncovalent bonds•  site of attack is amino acid group that participates in the normal enzymatic reactionReversible inhibition•  forms weak, noncovalent bonds that readily dissociate from an enzyme•  the enzyme is only inactive when the inhibitor is present
  • 96. Enzyme inhibition Competitive inhibitor •  resembles the normal substrate and competes for the same site
  • 97. Enzyme inhibition Examples of competitive inhibitors: •  methanol and ethylene glycol compete with ethanol for the binding sites to alcohol dehydrogenase •  methotrexate competes with folic acid for dihydrofolate reductase
  • 98. Enzyme inhibition Noncompetitive inhibitor •  materials that bind at a location other than the normal site •  results in a change in how the enzyme performs
  • 99. Enzyme inhibition Examples of noncompetitive inhibitors: •  physostigmine is a cholinesterase inhibitor used in the treatment of glaucoma •  captopril is an ACE inhibitor used in treatment of hypertension •  allopurinol is a xanthine oxidase inhibitor used to treat gout
  • 100. Enzyme inhibition Irreversible inhibitors •  permanently inactivate enzymes •  heavy metals (Hg2+, Pb2+, Cd2+) •  aspirin acetylates •  fluorouracil •  organophosphates
  • 101. Enzyme Inhibition - SummaryCompetitive•  Inhibitor binds at substrate site, inhibition is reversible as higher substratecompetes for inhibitor, Vmax unchanged, Km increasedNoncompetitive•  Inhibitor binds at site other than substrate, ESI cannot form product, increasedsubstrate does not compete, Km unchanged, Vmax decreased
  • 102. Competitive Inhibition
  • 103. Uncompetitive Inhibition
  • 104. Noncompetetive model
  • 105. Enzyme Regulation•  Proteolytic cleavage to activate: Enzyme exists in inactive form (zymogen) that isactivated by removal of a short peptide segment ( truncation)•  Covalent modification to increase or decrease activity, most common is phosphorylation•  Sequestration: enzyme forms inactive polymers•  Allosteric (“other site”) regulation, both positive and negative ( homotropic, heterotropic)Induction-upregulation: increase gene expression, synthesis of more enzymemoleculesRepression-downregulation: decrease gene expression, decrease synthesis ofenzyme molecules.
  • 106. Allosteric enzymesAre regulated by molecules called effectors(modifiers) that bind non-covalently at a siteother than active site. They can alter Vmax orKm or both)1. Homotrophic effectors – when the substrateitself is an effector2. Heterotrophic effector – when the effector isdifferent from a substrate (often it is an end-product - feedback inhibition)
  • 107. Allosteric enzymes show sigmoid curve(cooperative substrate binding like in Hb)
  • 108. Feedback inhibition
  • 109. Enzymes Used in Clinical diagnosesTissue damage: Increased release of tissue enzymes in plasma Enzyme assay is used for both diagnostic and prognostic purpose Eg: ALT – present in the liver will be appearing in the plasma if there is Liver damage or cell necrosis Isoenzymes: Structurally different enzymes but catalyze the same reaction Eg: CK1, CK2, CK3 (creatine kinase, CK MB (CK 2) is present in the heart, its presence in plasma is indicative of myocardial infarction
  • 110. ALSO: Troponin T & Troponin Iare also released in cardiacdamage. Peaks in 8 – 24hrSensitive and specific for cardiactissue damage

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