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ENZYMES

ENZYMES. Enzymes are proteins. Cofactors. Prostetic groups Metal ions Coenzymes. Covalent bond. Coordinative bond. Secondary interactions. Native conformation. Optimal Conditions :. pH. Ionic strength. Temperature. Solvent. Maximal catalytic activity. Active center:. Asp b COOH.

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ENZYMES

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  1. ENZYMES

  2. Enzymes are proteins Cofactors Prostetic groups Metal ions Coenzymes Covalent bond Coordinative bond Secondary interactions

  3. Native conformation Optimal Conditions: pH Ionic strength Temperature Solvent Maximal catalytic activity

  4. Active center: Asp bCOOH pKa = 4.5 - Lysozyme Glu gCOOH pKa = 5.9 pH 5 6 4 4.5 5.9 pH dependence of enzyme activity Asp - Glu Example: Optimal pH? deprotonated protonated Hw: Glu-, His+ (pKa 6.5) optimal pH

  5. Enzyme activity pH 4 5 6 = pH profiles Isoelectric pont pH optimum

  6. Optimal temperature Enzyme activity Q10 temperature coefficient T Temperature dependence of enzyme activity Heat denaturation changes in conformation Heat sensitivity is different Heat-shock proteins

  7. - D[S] Enzyme activity (EA) EA = EA = Dt + D[P] Dt Calculation of enzyme activity: substrate (S)  product (P) U = mmol/min U (unit) S.I. unit: Catal = mol/sec

  8. mol (product) mol (enzyme) x sec Turnover number (Molecular activity) examples Carbonic anhydrase: 400 000/sec Chymotrypsin: 100/sec Trp synthase: 2/sec

  9. Substrate (S) Product (P) S ES*1 ES *2 P ES* : enzyme-substrate complex G Eact S DG ES*2 ES*1 P Enzymes as catalysts

  10. S S* P E E ES*2 ES*1 E.g.: „Stickase”

  11. Effect of enzymes : Activation energy decreases DG (Kes ) does not change changes The reaction mechanism does not change The equilibrium state decreases The time to reach the EQ Enzymes are catalizing directions in both

  12. The enzyme - substrate complex Interactions: secondary bonds ionic bonds covalent bond (covalent catalysis) Acid-base catalysis: proton shuttle

  13. Enolase and 2-phospho-glycerate (2-PGA): ionic bond 2 Mg2+ cofactors

  14. S S E E Enzymes are specific catalysts The enzyme-substrate complex (ES) Lock and key model „induced fit” model

  15. Hexokinase induced-fit

  16. Hexokinase induced-fit

  17. S1 S2 E Entropy effect • Binding of substrates • close in space • proper orientation

  18. Dihydrofolate reductase NADP+ cofaktor dihidrofolate

  19. Dihydrofolate reductase NADP+ cofaktor dihidrofolate

  20. Medical-diagnostic significance of enzyme activity measurements 1. Non-functional plasma enzymes accelerated death of certain tissues soluble enzymes enter the circulation

  21. Aminotransferases: a reversible exchange between an a-amino and an a-keto group ALAT: alanine-amino transferase (or SGPT: serum glutamate - pyruvate aminotranspherase) pyruvate alanine a-ketoglutarate glutamate high ASAT in serum  hepatocellular tissue destruction e.g. in acute hepatitis (viral infection) in liver chirrosis in mononucleosis

  22. high ALAT in serum  muscle tissue destruction e.g. in myocardial infarction in acute rheumatoid carditis in the first 10 days of heart surgery after heart massage after catheter treatment of heart ASAT:aspartate-amino transferase (or SGOT: serum glutamate -oxaloacetate aminotranspherase) oxaloacetate aspartate a-ketoglutarate glutamate

  23. creatine creatine-P ADP ATP high MB in serum  myocardial disease e.g. in myocardial infarction (5-6 hrs after heart attack maximum value at 12 hrs) colon cancer  high MM renal failure  high MM Creatine kinase M: muscle form, B: brain form MM, MB or BB izoenzymes MB form is found exclusively in the heart

  24. Medical-diagnostic significance of enzyme activity measurements 2. Functional plasma enzymes

  25. Deficiency: cholesterol cannot be transported in the blood artheriosceloris, heart attack LCAT: lecitine-cholesterol acyl transferase free cholesterol cholesterol ester lysolecitine lecitine

  26. Medical-diagnostic significance of enzyme activity measurements 3. Enzyme activity assay in tissue biopsy

  27. pyruvate oxaloacetate ADP ATP, CO2 Deficiency: mental retardation Assay from skin biopsy Treatment: oral Asp, Glu Pyruvate carboxylase

  28. Enzymes as Catalysts Ser Proteases

  29. Acid-Base Catalysis A general acid His+ Cys-SH Tyr-OH Lys+ A general base His Cys-S - Tyr-O - Asp-, Glu -

  30. Covalent Catalysis E + S  [ES] E + P Covalent bond in the enzyme-substrate complex

  31. Ser Proteases • Protease: cuts proteins • hydrolysis of peptide bond • Ser-proteases: Ser in the active center • e.g. • Chymotrypsin • Trypsin • Ellastase • Thrombin • Other blood coagulation factors

  32. O O P F O Irreversible inhibition of Ser- enzymes DIPF: Specific inhibitor of Ser enzymes Di-isopropyl-fluoro-phosphate E Ser OH • e.g. • Chymotripsin • Acetylcholinesterase • (chemical weapon)

  33. Proteolytic activation of zymogens Intestine Pancreas Enteropeptidase Trypsinogen Chymotrypsinogen Trypsin p - Chymotrypsin a - Chymotrypsin Trypsin inhibitor Inactive trypsine

  34. H3N+ Chymotrypsin 15Arg 16Ile a-chimotrypsin(ACTIVE) Proteolytic activation of Chymotrypsin Chymotrypsinogen Trypsin p -chymotrypsin (ACTIVE) Interaction of 1NH3+ with 194Asp- Formation of substrate binding pocket

  35. 57His HN N 195 Ser 102 Asp HO Chymotrypsin: Acid-base catalysis The catalytic triad A proton shuttle in the active center

  36. O C Ser - O Ser - O Ser - OH NH His HisH+ His O- C NH O C H2N 1st product Chymotrypsin: Covalent catalysis Peptide bond of the food protein to be cut 1st tetrahedral transition intermedier Acyl- enzyme intermedier +

  37. O- Ser - O Ser - OH OH C HisH+ His O OH C 2nd product + H2O 2nd tetrahedral transition intermedier +

  38. R CH Substrate binding pocket O C NH Specificity of Ser proteases Chymotrypsin: Big, nonpolar pocket R: aromatic rings (Phe, Tyr, Trp) Trypsin: negatively charged pocket R: positive charges (Arg,Lys) Elastase: small pocket R: small (Gly,Ala, Ser)

  39. fibrinogen Proteolytic cleveage (Xa) OH A S B S thrombin fibrin prothrombin Thrombin is a Serine protease

  40. Enzyme Kinetics

  41. v1 P S v -1 1. v1 >>>v -1 D[S]  0 2. Enzyme activity (v) should be measured as initial rate: Conditions for measuring initial rate: [P]  0

  42. [P] time S P S P S P linearity equilibrium Initial rate v = constant v decreases Product formation as the function of the time [S] decreases [P] increases v = 0

  43. steady state The Michaelis-Menten model Assumptions: k1 k2 [ES] E + S E + P k - 1 k -2  0 IRREVERSIBLE [S]  [ES] One-substrate reaction Initial rate

  44. k1 k2 [ES] E + S E + P k - 1 steady state vmax [S] v = KM + [S] Rate of [ES] formation= Rate of [ES] breakdown Definition: (k2+k-1)/k1= KM

  45. v vmax 1 vmax 2 [S] KM I. Low [S] II. [S] = KM III. High [S] Enzyme activity (v, initial rates) as the function of substrate concentration Vmax = max EA KM = [S] where v = 1/2 vmax

  46. vmax [S] v = KM vmax [S] vmax [S] v = v = KM + [S] KM + [S] I. Low [S] II. [S] = KM III. High [S] First order reaction Michaelis-Menten Kinetics v = vmax saturated substrate concentration Zero order reaction

  47. Vmax kcat = [E] KM Michaelis constant KM = k-1 + k2 k1 kcat KM Turnover number kcat Affinity of the substrate to the enzyme low KM - high affinity „catalytic efficiency”

  48. 1/v 1/vmax 1/[S] - 1/KM Linearization for determination of KM and vmax Lineweaver-Burk double reciprocal plot Slope : Km/Vmax

  49. KM [S] 200 20 2 0.2 0.15 0.013 v 60 60 60 48 45 12 Vmax = 60 Vmax/2=30 60*0.013 12 = vmax [S] KM + 0.013 v = KM + [S] 9.1.2.1. Calculate the KM and the vmaxfrom the data below!

  50. [S] 1/[S] 200 20 2 0.2 0.15 0.013 1/200 and so on v 1/v 60 60 60 48 45 12 1/60 and so on Calculate the reciprocal values! 9.1.2.1. Calculate the Vmax and KM using a graph!

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