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

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

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: + + +

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

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)

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

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

35. Reciprocal equation

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

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

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

52. Example of i rreversible inhibition Diisopropyl fluorophosphate (and similar pesticides and nerve gases) inhibits acetylcholine esterase by phosphorylation of a crucial serine residue.

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

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

68. Allosteric enzymes are oligomeric

69. Satura tion curve of allosteric enzym es is sigmoid al bez efektoru activation without effector inhibition

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

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)