1. Lecture 6: Kumar Measuring enzyme activity 1

2. Effect of pH on enzyme activity 2

3. 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 Effect of temperature on enzyme activity 3

4. 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) 5

6. MICHAELIS-MENTEN EQUATION Based upon the formation of ES complex, following equation is derived V max [S] v = Km + S V max 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

7. Km and Vmax 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]

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

11. PHYSIOLOGICAL IMPORTANCE OF Km IN GLUCOSE HOMEOSTASIS 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?

12. Treatment of Leukemia 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 SourceKm (uM)VmaxVmax/Km Bovine200500.25 Rat40300.75 Bacteria 13030 The ratio Vmax/Km is referred to as efficiency of catalysis.

13. Lecture 6-Measurement of Enzyme Activity 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?