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513340 Basic Biochemistry

513340 Basic Biochemistry. ENZYME KINETICS Porntip Chaimanee Chemistry Dept, Faculty of Science Silpakorn University. Enzymatic Analysis. Enzyme (E). Time. Temperature. Substrate (S). Product (P). อัตราเร็วของปฎิกิริยา (velocity, v) = d[P]/dt หรือ -d[S]/dt.

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513340 Basic Biochemistry

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  1. 513340 Basic Biochemistry ENZYME KINETICS PorntipChaimanee Chemistry Dept, Faculty of Science Silpakorn University

  2. Enzymatic Analysis Enzyme (E) Time Temperature Substrate (S) Product (P) อัตราเร็วของปฎิกิริยา(velocity, v) = d[P]/dt หรือ -d[S]/dt Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  3. Principle of spectrophotometry L Detector Monochromator I I0 Absorption recorder Light source Sample Absorption A = -log I/Io = e.c.L Beer-Lambert law Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  4. Assay of enzyme activity Time Absorbance (sec) of product 15 0.075 30 0.15 45 0.20 60 0.26 75 0.32 90 0.36 105 0.40 120 0.44 150 0.51 180 0.57 210 0.62 240 0.66 E Absorption Time (sec) Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  5. AP Reaction velocity, rate of reaction, v v = -d[A ]/dt =d[P]/dt v = k[A] k, rate constant (rate coefficient, specific reaction rate) Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  6. Velocity or Rate determination Initial rate (slope at t = 0) [Product] Concentration Velocity (vO) = slope at t = t1 [Substrate] t = t1 t = 0 Time Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  7. 1902 Adrian Brown Experiment Sucrose + H2O glucose + fructose yeast enzyme (invertase) Reaction rate become independent to [Sucrose], when [Sucrose] >>> [Invertase enzyme] Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  8. 1913 Michaelis Menten propose Initial Rate , Steady state conditions k1 k2 k-1 E + S ES P + E E = free enzyme ES = enzyme-substrate complex k1, k-1,k2 : rate constants (S) >>> [E] + [ES] Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  9. k1 k2 k-1 E + S ES E + P 1 2 1 Presteady state conditions, a few milliseconds 2 Steady state conditions : a few minutes Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  10. Dissociation constant of ES Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  11. ET= E+ ES KS = + k2/k1 Michaelis constant Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  12. =kcat[E]T Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  13. Vmax =k2[E]T =kcat[E]T kcat = Vmax [E]T kcat(turnover number) = maximum number of substrate turn into product in one second vO=Vmax [S] KM +[S] at physiological condition [S] <<< KM = Vmax KM [S] = (kcat/KM) [E]T[S] Specificity constant How rapidly an enzyme can work at low [S]

  14. The values of kcat ,KMand(kcat/KM) for some enzymes and substrates Enzyme Substrate kcat (s-1) KM (M) kcat/KM(M-1 s-1) Acetylcholine esterase Acetylcholine 1.4x 104 9.5x 10-5 1.5x 108 Carbonic anhydrase CO2 1.0x 106 1.2x 10-2 8.3x 107 HCO3 4.0x 105 2.6x 10-2 1.5x 107 Catalase H2O2 1.0x 107 2.5x 10-2 4.0x 108 Fumarase Fumarate 8.0x 102 5.0x 10-6 4.6x 108 Malate 9.0x 102 2.5x 10-5 3.6x 107 Chymotrypsin N-Acetylglycine ethyl ester 5.1x 10-2 4.4x 10-1 1.2x 10-1 N-Acetylvaline ethyl ester 1.7x 10-1 8.8x 10-2 1.9 N-Acetyltyrosine ethyl ester 1.9x 102 6.6x 10-4 2.9x 105

  15. Variation of initial rate (v0) as a function of [S]with constant concentration of enzyme In blue : v = d[P]/dt = constant [P] Increasing [S] time Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  16. Variation of initial rate(v0) as a function of [S]with constant concentration of enzyme = E = S Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  17. The Lineweaver-Burk plot y = m.x + b Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  18. A double-reciprocal (Lineweaver-Burk) plot. Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  19. ENZYME INHIBITION การทำงานของเอนไซม์อาจถูก ยับยั้งหรือรบกวน โดยสารบางชนิดที่สามารถ ยื้อแย่ง กับ substrate ในการเข้าจับกับเอนไซม์ทำให้ประสิทธิภาพ ในการทำงานลดลงหรือเสียไป Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  20. TWO TYPES OF INHIBITORS 1. IRREVERSIBLE INHIBITORS 2. REVERSIBLE INHIBITORS Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  21. IRREVERSIBLE INHIBITORSCompounds Reactive group of enzymes Examples CN- enzyme metal ions ( eg. Fe, Zn ,Cu ) O Sarin (CH3)2-CH-O-P-CH3 -SH group of cysteine Acetylcholine esterase O F Prathion C2H5O-P-O- -NO2 -SH group of enzyme Acetylcholine esterase F O DIPFP (CH3)-CH-O-P-O-CH-CH3 -OH group of serine Acetylcholine esterase Nerve gas CH3 F CH3 Chymotrypsin Iodoacetamide I-CH2-C-NH2 -SH group of cysteine Glyceraldehyde-3- O phosphate dehydrogenase Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  22. Penicillin is an irreversible of the enzyme glycoprotein peptidase

  23. HIV PROTEASE INHIBITORS -Phe-Phe- HIV protease substrate Saquinavir KI=0.40 nM Ritonavir KI=0.015 nM

  24. X-Ray structure of HIV-1 protease Asp (a) Uncomplexed (b) in complexed with its inhibitor saquinavir

  25. REVERSIBLE INHIBITORS k1 k2 k-1 E + S ES E + P General principle: a) Any inhibitor that displaces the equilibrium E + S ES modifies the Km b) Any inhibitor that changes the maximum concentration of ES modifies the Vmax Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  26. Competitive Inhibitors Substrate Enzyme k1 k2 k-1 E + S ES P + E + I EI + S NO REACTION P ES complex KI Competive inhibitor Inactive enz EI complex Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  27. Variation of initial rate(v0) as a function of [S]with and without competitive inhibitor Vmax Uninhibited v0 Competitive inhibition Vmax/2 KM aKM [S] Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  28. Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  29. A double-reciprocal (Lineweaver-Burk) plot with and without competitive inhibitor KM increased Vmaxunchanged KM increased Vmaxunchanged Increasing [I] 1/vo Uninhibited -1/KM 1/[S] -1/aKM Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  30. Noncompetitive Inhibitors Substrate Enz k1 k2 k-1 E + S ES P + E + + I I EI + S ESI NO REACTION P ES complex Noncompetitive inhibitor KI K/ I Inactive enz Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  31. Variation of initial rate(v0) as a function of [S]with and without noncompetitive inhibitor Vmax Uninhibited v0 Vmax/2 Noncompetitive inhibition KM [S] Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  32. A double-reciprocal (Lineweaver-Burk) plot with and without noncompetitive inhibitor KM unchanged Vmaxdiminished Increasing [I] 1/vo 1/aVmax Uninhibited 1/Vmax -1/KM 1/[S] Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  33. Uncompetitive Inhibitors Substrate Enz k1 k2 k-1 E + S ES P + E + I ESI NO REACTION P ES complex K/ I Uncompetitive inhibitor Inactive enz Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  34. Variation of initial rate(v0) as a function of [S]with and without uncompetitive inhibitor Uninhibited v0 Uncompetitive inhibition aKM KM [S] Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  35. A double-reciprocal (Lineweaver-Burk) plot with and without uncompetitive inhibitor KM diminished Vmaxdiminished Slope= KM /Vmax 1/vo Increasing [I] 1/aVmax Uninhibited 1/Vmax -1/KM 1/[S] -1/aKM Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  36. Effect of temperature on velocity vo Arrhenius / Law Thermal denaturation Optimum temperature Temperature Chemistry Science Silpakorn University Dr. Porntip Chaimanee

  37. Effect of pH on velocity Pepsin vo Trypsin pH 1.5 7.7 Optimum pH Chemistry Science Silpakorn University Dr. Porntip Chaimanee

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