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Lecture 6: Measuring enzyme activity

Lecture 6: Measuring enzyme activity. Effect of pH on enzyme activity. Effect of temperature on enzyme activity. Curve blue: Enzyme isolated from shrimp in cold waters of Alaska Curve red: Physiological temp working enzymes-porcine chymotrypsin

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Lecture 6: Measuring enzyme activity

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  1. Lecture 6: Measuring enzyme activity

  2. Effect of pH on enzyme activity

  3. Effect of temperature on enzyme activity Curve blue: Enzyme isolated from shrimp in cold waters of Alaska Curve red: Physiological temp working enzymes-porcine chymotrypsin Curve green: enzymes isolated from bacteria living in thermal springs

  4. ENZYMES-Activity Measurements Enzyme and substrate form a complex k1 k3 E + S ES E + P k2

  5. Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v)

  6. Based upon the formation of ES complex, following equation is derived Vmax [S] v = Km + S Vmax is maximal velocity when enzyme is saturated with the substrate. Km is Michaelis constant and in many cases is equal to the dissociation constant of ES complex MICHAELIS-MENTEN EQUATION

  7. Km Is a ratio of rate constants =(k2 + k3)/k1 Is equal to [S] when initial rate(v) is equal to ½ Vmax Is a property of ES complex; does not depend on the concentration of E or S Vmax Maximum velocity at a fixed E concentration Directly proportional to the [E] Km and Vmax

  8. First and Zero order Enzyme Kinetics Vmax x[S] Rate (v) = Km + [S] When [S] is << Km v= Vmax x [S]/Km The reaction is first order wrt [S]. This ratio gives the efficiency of catalysis When [S] is >>> Km; then MM equation is reduced to: V= Vmax so the rate is independent of [S] and this is called zero order rate.

  9. Lineweaver-Burk Double reciprocal Plot

  10. Vmax directly proportional to [E]; so make activity measurements at saturating [S]. To do so have S~ 10Km And then v= 10/11 Vmax MEASUREMENT OF ACTIVITY IN CLINICAL SAMPLES AND FLUIDS (10/11)Vmax [E]

  11. Plasma [Glucose]= 100 mg% ~ 5 mM Brain Glucose = 2 mM Km for glucose in liver for glucokinase = 15 mM Km for glucose in brain for hexokinase = 0.01 mM Km for ATP for both liver and brain enzymes = 0.1 mM Glucose + ATP Glucose-6-phosphate + ADP At 1.0 mM ATP in cells, glucose will be oxidized in liver at 25 % of Vmax In brain at ~100 % of Vmax Please Calculate? PHYSIOLOGICAL IMPORTANCE OF Km IN GLUCOSE HOMEOSTASIS

  12. PML patients have high plasma Asparagine(Asn) Concentration (50 uM). The objective is to lower this concentration to 5 uM. Asn + H2O -------------> Asp + NH3 asparaginase Source Km (uM) Vmax Vmax/Km Bovine 200 50 0.25 Rat 40 30 0.75 Bacteria 1 30 30 The ratio Vmax/Km is referred to as efficiency of catalysis. Treatment of Leukemia

  13. Learning Objectives: How do enzyme catalyzed reactions differ from other catalyzed reaction? What are the assumptions in the derivation and what is the significance of MM equation? How can one determine Km and Vmax? What is the significance of Km and Vmax? What conditions are used in clinical analyses? How can the knowledge of Km values be used to explain physiological function? Is the knowledge of Km values useful in designing drugs? Lecture 6-Enzyme Kinetics

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