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Enzymes II    Department of  Bioche mistry (J.D.) 2011 Mechanism  of  enzym e  rea ction , metal l oenzym es , kineti cs , a c tivit y , enzym es in medicine
Active site of enzyme ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Binding substrate to  enzym e ,[object Object],[object Object]
Example:  The active sites of three proteinases Certain  AA  residues determin e  the substrate specificity of these enzymes. The peptide bonds prefer ably hydrolyzed:   by  chymotrypsin  -  near  residues with large, hydrophobic, uncharged side chains (Phe, Trp), by  trypsin  –   near  residues with long, positively charged side chains (Arg, Lys),  by  elastase   - near  residues with small hydrophobic side chains (Gly, Ala). A deep, relatively hydrophobic pocket At the bottom of the deep pocket there is an acidic residue (Asp) Two residues of valine close off the mouth of the small hydrophobic pocket
C atalytic   mechanism  depends on the number of  substr ates ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Sequential reaction : -  type  ordered  -  the substrates bind the enzyme in a defined sequence -  type  random   – the order of addition of substrates and release of products is random: Ping-pong reaction: + + +
Catalytic groups  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
CYSTEINE   PROTEINASES Carboxypeptidase A, thermolysin (bacterial) METALLOPROTEINASES Cathepsins, papain  ASPART ATE  PROTEINASES Pepsin, renin Example s of  proteinases ,[object Object],[object Object],[object Object],SERINE   PROTEASES Serine 195 Chymotrypsin , trypsin
Example: active site of chymotrypsin Nucleophilic attack of serine -OH to carbonyl carbon of peptide bond      serine protease 195 57 simplified
Active site of chymotrypsin:  the cleavage of peptide bond
Metalloenzymes ,[object Object],[object Object]
Metal ion is a part of ternary complex ,[object Object],[object Object],[object Object],[object Object],[object Object],possible   mechanism
Molybdenum (Mo) ,[object Object],[object Object],[object Object],[object Object],Mo in food: legumes, wholemeal cereals
Zinc (Zn) ,[object Object],[object Object],[object Object],[object Object],[object Object],Zn in food: red meat, lobsters, legumes, seeds, wholemeal cereals
Copper (Cu) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Cu in food: liver, meat, cocoa, legumes, nuts
Manganese (Mn) ,[object Object],[object Object],[object Object],[object Object],Mn in food: legumes, nuts, wholemeal cereals
Iron (Fe) ,[object Object],[object Object],[object Object],[object Object],[object Object],Fe in food: animal blood, red meat, liver, egg yolk, nuts, broccoli
Selenium (Se) ,[object Object],[object Object],[object Object],[object Object],Se in food: sea products, legumes
Basic  kinetic  terms ,[object Object],[object Object],this definiton is for average rate, instantaneous rate:  d[S]/d t
Factors influencing  rea ction rate  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Kinetic  equation for  rea ction  S    P  v   =  k  [S]  =  k [S] 1      reaction of 1. order k =  rate   c onstant
K inetic   (progress)  curve s  for  substrate and  product during  rea ction : concentra tion of  substr ate  decreases concentration of product increases reaction rate is determined from kinetic curves The instantaneous velocity  v x  at any particular time  t x  is  g iven  by the slope of the tangent to the curve at that time. [ P ] t [ S ] t (equilibrium)
Rea ction of  0.  order  ,[object Object],[object Object],[object Object],in enzyme rections only in  laboratory conditions
Initial velocity   v o ,[object Object],[object Object],[object Object]
Initial velocity  v o  depends on  substr ate concentration ,[object Object],[object Object],V max  = maximal velocity  (for the given concentration of enzyme) K m  = Michaelis constant
The graph of previous equation is saturation curve enzyme saturated by substrate
If  [S] <<  K m at low substrate concentration the reaction proceeds by the  1 st  order kinetics ×
If  [S] >>  K m at high substrate concentration the reaction proceeds by the  0. order kinetics ×
Two parts of saturation curve compare with   pages 21 and 23 [ S ] V 0 V max K m The zero-order kinetics The 1 st  order kinetics
If  [S] =  K m
Significance of  K m  and   V max   ,[object Object],[object Object],[object Object],[object Object]
From the obtained progress curves, the values  v o   are   estimated   and plotted against  the corresponding [S] .  T he velocity  v o   rises linearly as substrate concentration increases, and then begins to level till it reaches a limit value at high substrate concentrations. How to get a saturation curve?  E   = const . A series of measurements of initial velocity  is  arranged   at a constant enzyme concentration   E   and different substrate   concentrations   S   (in the range of 2 - 3 orders).  [ P ] t [ S 1 ] [ S 2 ] [ S 3 ] [ S 4 ] v 0  1 v 0  2 v 0  3 v 0  4
Distinguish ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[S] ……..  substr ate concentration [P]  ..........  product concentration f …………  function t  …………  time v o   ………..  initial velocity
V max  and   K m  describe the kinetic properties  of enzyme ,[object Object],[object Object],[object Object]
Reciprocal equation
Reciprocal form is the equation of a line  ( y  =  a  x   +  b) 1/ v o   ................  dependent variable ( y ) 1/ [S]  ...............  independent variable ( x ) 1/ K m  ........... ....   1/ V max   .............  easily determined from the graph
Linear reciprocal plot: 1/ v o  is the function of 1/ [S]
Initial velocity depends on  enzym e concentration  [E]  v o [S] [E 1 ] [E 2 ]  >  [E 1 ] [E 3 ]  >  [E 2 ] K M   does not change,  V max   increases with increased  [E] saturated  enzym e :  v o   =  k [E] t   [E] t   is  tot al c oncentra tion of  enzym e
How to quantify  enzym es  in b iologic al  mater ial ? ,[object Object],[object Object],[object Object],[object Object],[object Object]
Enzyme quantity in a biological sample  can be determined by two ways  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Catalytic  activity  of enzyme ,[object Object],[object Object],IU (international unit), 1 IU =  μ mol/min 1  μ kat = 60 IU,  1 IU = 16.6 nkat Catalytic  concentration  of enzyme ,[object Object],[object Object]
Distinguish: unit and its dimension mol/l.s kat/l Catalytic concentration mol/s kat Catalytic activity Dimension Unit Quantity
Determination of catalytic activity of ALT:  two coupled enzyme reactions alanine + 2-oxoglutarate     pyruvate  +  glutamate blood serum sample (ALT) pyridoxal-P pyruvate + NAHD+H +      lactate  + NAD + Δ A/ Δ t optical (UV) test Semin ars ,  p .18 the decrease of absorbance at 340 nm is followed in time LD
Determination of catalytic activity   in vitro ,[object Object],[object Object],[object Object]
Two methods for catalytic concentration less exact exact  Evaluation of method average velocity initial velocity  v o What is determined no yes Kinetic curve needed after some time (e.g. 10 min) the reaction is stopped  by enzyme inactivation continually  ( e.g. in  10 s econds ) How [P]  [S]  or [P]  What is measured Constant-time method Kinetic method Feature
Problem 1 Enzyme sample (0.1 ml) was added to substrate solution.  After 5 min,  0 . 2 mmol  of  produ ct was determined. What is catalytic concentration  of enzyme?
Solution t   = 5 min = 5  ×  60 s = 300 s in 300 s …  0.2 mmol of product in  1 s … x = 0.2/300  =  6.7  × 10 -4  mmol / 0.1 ml of sample for 1 litre of sample = 6.7  ×  10 -4   ×  10 4  = 6.7 mmol/l.s  =  6.7 mkat/l
Problem 2 Reaction mixture contains:  2.5 ml buffer  0.2 ml solution of NADH (optical UV test) 0.1 ml blood serum  0.2 ml substrate solution After 60 s, the decrease of NADH absorbance is   A  = 0.03   NADH   =  6220 l/mol.cm, cuvette width  l  = 1 cm.  What is catalytic concentration of enzyme?
Solution Serum sample was diluted: V final /V initial  =  3,0 / 0,1  =  30 Lambert-Beer law:   A   =      c   l   changes of absorbance and concentration expressed per time   t    A /  t   =      c   l /  t   t   = 60 s Multiplied by dilution:  30  ×  8  ×  10 -8   =  2,4  ×  10 -6  mol/l.s  =  2,4  ×  10 -6  kat/l =  2,4   kat/l
Consider that  catalytic concentration  =  rate of chemical reaction kat /l   =  mol/l.s
[object Object],[object Object],[object Object],[object Object],[object Object],Inhibitors reduce enzyme activity Reversible inhibit ors In contrast with irreversible inhibitors, reversible inhibitors bind to the enzyme loosely and can rapidly dissociate from the enzyme-inhibitor complex.  These inhibitors are classified as  competitive ,  non-competitive .
Example of i rreversible inhibition Diisopropyl   fluorophosphate (and similar pesticides and nerve gases) inhibits acetylcholine esterase by phosphorylation of a crucial serine residue.
Competitive inhibitors ,[object Object],[object Object],[object Object]
Malonate competitively inhibit succinate dehydrogenase Examples: Methotrexate competitively inhibits active sites for tetrahydrofolate of the dihydrofolate reductase in the synthesis of purine and pyrimidine bases of nucleic acids. It is used to treat cancer. Methotrexate Tetrahydrofolate Malonate Succinate COO – CH 2 CH 2 COO – COO – CH 2 COO –
Methanol poisoning is treated by ethanol ethanol and methanol are similar molecules they compete for active site in enzyme alcohol dehydrogenase
Competitive inhibitors increase  K m   without any change in  V max   The  V max  can be reached even in the presence of inhibitor, but at much higher concentrations of  [S] that have to overcome the competing inhibitor concentration. K m [S] v 0 V max K m 1 /  v 0 1 / [S] 1 /  V max - 1 /  K m No inhibitor Competitive inhibitor
Non - competitive inhibition Non - competitive inhibitors bind to both free enzyme and enzyme-substrate complex, but in contrast to competitive inhibitors,  not in   the active site   (the structure of inhibitor is distinct from th at of  substrate ). Non - competitive inhibition cannot be overcome by increasing the substrate concentration. The non - inhibited remaining molecules of the enzyme behave like a  more diluted solution of the enzyme.
Non - competitive inhibitors decrease  V max  without any change in  K m 1/v o 1 / [S] 1 /  V max - 1 /  K m Non - competitive inhibitor No inhibitor K m [S] v 0 V max V max Non - competitive inhibitor
Many drugs are inhibitors of enzymes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],Regulation principles in enzyme reactions  (three general aspects)
Regulation of enzyme quantity ,[object Object],[object Object]
Regulation of enzyme activity ,[object Object],[object Object],[object Object]
Enzyme activation by partial proteolysis ,[object Object],[object Object],[object Object],[object Object]
Reversible covalent enzyme modification ,[object Object],[object Object],[object Object],[object Object]
Phosphorylation and dephosphorylation  of enzyme   alters its  a c tivit y kinase phosphatase
The consequences of phosphorylation/dephosphorylation  in two antagonistic enzymes activated inhibited By dephosphorylation, enzyme is inhibited activated By phosphorylation, enzyme is glycogen synthesis  from energy-rich  UDP-glucose  glycogen degradation  by energy-poor  phosphate (P i ) Enzyme catalyzes Glycogen synthase Glycogen phosphorylase Feature
Allosteric enzymes are oligomeric ,[object Object],[object Object],[object Object]
Allosteric enzymes are oligomeric
Satura tion curve of  allosteric   enzym es is  sigmoid al bez  efektoru activation without  effector inhibition
Cooperative effect  ,[object Object],[object Object],[object Object],[object Object]
Three utilizations of enzymes in medicine ,[object Object],[object Object],[object Object]
Examples of enzymes in clinical diagnostics ALT alanine aminotransferase, CK creatine kinase,   PSA prostate specific antigen In cell damages, activity of  intracellular  enzymes in  extracellular  fluid (blood serum) is elevated hepatopaties myopaties, myocardial infarction prostate cancer Elevation in serum indicates up to 0,9   kat/l up to 4   kat/l up to  4   μ g/l ALT CK PSA Reference values Enzyme
Creatine kinase (CK) is a dimer and makes three isoenzymes ,[object Object],[object Object],[object Object],[object Object],Isoenzymes/Isoforms muscle trauma infarction  brain damage Elevated value 94-96 % up to 6 % traces muscles heart brain CK-MM CK-MB CK-BB % of total activity  in blood plasma  Organ Isoenzyme
Enzymes as analytic reagents glucose glucose triacylglycerols cholesterol uric acid bilirubin urea ALT, AST PCR met hod Aspergillus niger horse   radish  ( Armoracia  sp. ) Candida  sp. Pseudomonas  sp. Candida  sp. Myrothecium  sp. Canavalia  sp. Pediocus  sp. Thermus aquaticus Glucose oxidase Peroxidase Lipase Cholesterol oxidase Uricase Bilirubin oxidase Urease Lactate dehydrogenase Taq  polymeras e Assay for Enzyme origin Enzyme
Enzymatic determination of glucose glucose  +  O 2      gluconolactone  +  H 2 O 2 H 2 O 2   +  H 2 A     2 H 2 O  +  A glucose oxidase peroxidase colourless chromogen coloured product (absorbance measured) The principle of glucose determination in biochemical analyzers and in personal glucometers
Personal glucometer  (Practice in 4. semester)
Pancreatic enzymes in therapy ,[object Object],[object Object],[object Object],acidoresistant capsules, they survive the passage through stomach, soluble till in duodenum
Asparaginase in leukemia treatment ,[object Object],[object Object],[object Object],[object Object],Enzyme fibrinolytics ,[object Object],[object Object],[object Object],[object Object]
Proteases in enzyme therapy ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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Enzymes 2

  • 1. Enzymes II  Department of Bioche mistry (J.D.) 2011 Mechanism of enzym e rea ction , metal l oenzym es , kineti cs , a c tivit y , enzym es in medicine
  • 2.
  • 3.
  • 4. Example: The active sites of three proteinases Certain AA residues determin e the substrate specificity of these enzymes. The peptide bonds prefer ably hydrolyzed: by chymotrypsin - near residues with large, hydrophobic, uncharged side chains (Phe, Trp), by trypsin – near residues with long, positively charged side chains (Arg, Lys), by elastase - near residues with small hydrophobic side chains (Gly, Ala). A deep, relatively hydrophobic pocket At the bottom of the deep pocket there is an acidic residue (Asp) Two residues of valine close off the mouth of the small hydrophobic pocket
  • 5.
  • 6. Sequential reaction : - type ordered - the substrates bind the enzyme in a defined sequence - type random – the order of addition of substrates and release of products is random: Ping-pong reaction: + + +
  • 7.
  • 8.
  • 9. Example: active site of chymotrypsin Nucleophilic attack of serine -OH to carbonyl carbon of peptide bond  serine protease 195 57 simplified
  • 10. Active site of chymotrypsin: the cleavage of peptide bond
  • 11.
  • 12.
  • 13.
  • 14.
  • 15.
  • 16.
  • 17.
  • 18.
  • 19.
  • 20.
  • 21. Kinetic equation for rea ction S  P v = k [S] = k [S] 1  reaction of 1. order k = rate c onstant
  • 22. K inetic (progress) curve s for substrate and product during rea ction : concentra tion of substr ate decreases concentration of product increases reaction rate is determined from kinetic curves The instantaneous velocity v x at any particular time t x is g iven by the slope of the tangent to the curve at that time. [ P ] t [ S ] t (equilibrium)
  • 23.
  • 24.
  • 25.
  • 26. The graph of previous equation is saturation curve enzyme saturated by substrate
  • 27. If [S] << K m at low substrate concentration the reaction proceeds by the 1 st order kinetics ×
  • 28. If [S] >> K m at high substrate concentration the reaction proceeds by the 0. order kinetics ×
  • 29. Two parts of saturation curve compare with pages 21 and 23 [ S ] V 0 V max K m The zero-order kinetics The 1 st order kinetics
  • 30. If [S] = K m
  • 31.
  • 32. From the obtained progress curves, the values v o are estimated and plotted against the corresponding [S] . T he velocity v o rises linearly as substrate concentration increases, and then begins to level till it reaches a limit value at high substrate concentrations. How to get a saturation curve?  E  = const . A series of measurements of initial velocity is arranged at a constant enzyme concentration  E  and different substrate concentrations  S  (in the range of 2 - 3 orders). [ P ] t [ S 1 ] [ S 2 ] [ S 3 ] [ S 4 ] v 0 1 v 0 2 v 0 3 v 0 4
  • 33.
  • 34.
  • 36. Reciprocal form is the equation of a line ( y = a x + b) 1/ v o ................ dependent variable ( y ) 1/ [S] ............... independent variable ( x ) 1/ K m ........... .... 1/ V max ............. easily determined from the graph
  • 37. Linear reciprocal plot: 1/ v o is the function of 1/ [S]
  • 38. Initial velocity depends on enzym e concentration [E] v o [S] [E 1 ] [E 2 ] > [E 1 ] [E 3 ] > [E 2 ] K M does not change, V max increases with increased [E] saturated enzym e : v o = k [E] t [E] t is tot al c oncentra tion of enzym e
  • 39.
  • 40.
  • 41.
  • 42. Distinguish: unit and its dimension mol/l.s kat/l Catalytic concentration mol/s kat Catalytic activity Dimension Unit Quantity
  • 43. Determination of catalytic activity of ALT: two coupled enzyme reactions alanine + 2-oxoglutarate  pyruvate + glutamate blood serum sample (ALT) pyridoxal-P pyruvate + NAHD+H +  lactate + NAD + Δ A/ Δ t optical (UV) test Semin ars , p .18 the decrease of absorbance at 340 nm is followed in time LD
  • 44.
  • 45. Two methods for catalytic concentration less exact exact Evaluation of method average velocity initial velocity v o What is determined no yes Kinetic curve needed after some time (e.g. 10 min) the reaction is stopped by enzyme inactivation continually ( e.g. in 10 s econds ) How [P] [S] or [P] What is measured Constant-time method Kinetic method Feature
  • 46. Problem 1 Enzyme sample (0.1 ml) was added to substrate solution. After 5 min, 0 . 2 mmol of produ ct was determined. What is catalytic concentration of enzyme?
  • 47. Solution t = 5 min = 5 × 60 s = 300 s in 300 s … 0.2 mmol of product in 1 s … x = 0.2/300 = 6.7 × 10 -4 mmol / 0.1 ml of sample for 1 litre of sample = 6.7 × 10 -4 × 10 4 = 6.7 mmol/l.s = 6.7 mkat/l
  • 48. Problem 2 Reaction mixture contains: 2.5 ml buffer 0.2 ml solution of NADH (optical UV test) 0.1 ml blood serum 0.2 ml substrate solution After 60 s, the decrease of NADH absorbance is  A = 0.03  NADH = 6220 l/mol.cm, cuvette width l = 1 cm. What is catalytic concentration of enzyme?
  • 49. Solution Serum sample was diluted: V final /V initial = 3,0 / 0,1 = 30 Lambert-Beer law:  A =   c l changes of absorbance and concentration expressed per time  t   A /  t =   c l /  t  t = 60 s Multiplied by dilution: 30 × 8 × 10 -8 = 2,4 × 10 -6 mol/l.s = 2,4 × 10 -6 kat/l = 2,4  kat/l
  • 50. Consider that catalytic concentration = rate of chemical reaction kat /l = mol/l.s
  • 51.
  • 52. Example of i rreversible inhibition Diisopropyl fluorophosphate (and similar pesticides and nerve gases) inhibits acetylcholine esterase by phosphorylation of a crucial serine residue.
  • 53.
  • 54. Malonate competitively inhibit succinate dehydrogenase Examples: Methotrexate competitively inhibits active sites for tetrahydrofolate of the dihydrofolate reductase in the synthesis of purine and pyrimidine bases of nucleic acids. It is used to treat cancer. Methotrexate Tetrahydrofolate Malonate Succinate COO – CH 2 CH 2 COO – COO – CH 2 COO –
  • 55. Methanol poisoning is treated by ethanol ethanol and methanol are similar molecules they compete for active site in enzyme alcohol dehydrogenase
  • 56. Competitive inhibitors increase K m without any change in V max The V max can be reached even in the presence of inhibitor, but at much higher concentrations of [S] that have to overcome the competing inhibitor concentration. K m [S] v 0 V max K m 1 / v 0 1 / [S] 1 / V max - 1 / K m No inhibitor Competitive inhibitor
  • 57. Non - competitive inhibition Non - competitive inhibitors bind to both free enzyme and enzyme-substrate complex, but in contrast to competitive inhibitors, not in the active site (the structure of inhibitor is distinct from th at of substrate ). Non - competitive inhibition cannot be overcome by increasing the substrate concentration. The non - inhibited remaining molecules of the enzyme behave like a more diluted solution of the enzyme.
  • 58. Non - competitive inhibitors decrease V max without any change in K m 1/v o 1 / [S] 1 / V max - 1 / K m Non - competitive inhibitor No inhibitor K m [S] v 0 V max V max Non - competitive inhibitor
  • 59.
  • 60.
  • 61.
  • 62.
  • 63.
  • 64.
  • 65. Phosphorylation and dephosphorylation of enzyme alters its a c tivit y kinase phosphatase
  • 66. The consequences of phosphorylation/dephosphorylation in two antagonistic enzymes activated inhibited By dephosphorylation, enzyme is inhibited activated By phosphorylation, enzyme is glycogen synthesis from energy-rich UDP-glucose glycogen degradation by energy-poor phosphate (P i ) Enzyme catalyzes Glycogen synthase Glycogen phosphorylase Feature
  • 67.
  • 69. Satura tion curve of allosteric enzym es is sigmoid al bez efektoru activation without effector inhibition
  • 70.
  • 71.
  • 72. Examples of enzymes in clinical diagnostics ALT alanine aminotransferase, CK creatine kinase, PSA prostate specific antigen In cell damages, activity of intracellular enzymes in extracellular fluid (blood serum) is elevated hepatopaties myopaties, myocardial infarction prostate cancer Elevation in serum indicates up to 0,9  kat/l up to 4  kat/l up to 4 μ g/l ALT CK PSA Reference values Enzyme
  • 73.
  • 74. Enzymes as analytic reagents glucose glucose triacylglycerols cholesterol uric acid bilirubin urea ALT, AST PCR met hod Aspergillus niger horse radish ( Armoracia sp. ) Candida sp. Pseudomonas sp. Candida sp. Myrothecium sp. Canavalia sp. Pediocus sp. Thermus aquaticus Glucose oxidase Peroxidase Lipase Cholesterol oxidase Uricase Bilirubin oxidase Urease Lactate dehydrogenase Taq polymeras e Assay for Enzyme origin Enzyme
  • 75. Enzymatic determination of glucose glucose + O 2  gluconolactone + H 2 O 2 H 2 O 2 + H 2 A  2 H 2 O + A glucose oxidase peroxidase colourless chromogen coloured product (absorbance measured) The principle of glucose determination in biochemical analyzers and in personal glucometers
  • 76. Personal glucometer (Practice in 4. semester)
  • 77.
  • 78.
  • 79.