Transcript Document
Lecture 6: Kumar Measuring enzyme activity 1 Effect of pH on enzyme activity 2 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 3 ENZYMES-Activity Measurements Enzyme and substrate form a complex k1 k3 E+S ES E + P k2 4 Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v) 5 MICHAELIS-MENTEN EQUATION • 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 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] 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. Lineweaver-Burk Double reciprocal Plot 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 [E] 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? 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 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. 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?