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3. E NZYMES o Enzyme reaction: E + S ↔ ES → E + P Whereas: E: Enzyme, S: Substrate, ES: Enzyme-Substrate complex, P: Product o Enzyme reaction: E + S ↔ ES → E + P Whereas: E: Enzyme, S: Substrate, ES: Enzyme-Substrate complex, P: Product 3

4. E NZYMES DECREASE ACTIVATION ENERGY A chemical reaction goes through a transition state with a higher  G than either S of P Enzymes facilitate the formation of the transition state by decreasing  G ‡ A chemical reaction goes through a transition state with a higher  G than either S of P Enzymes facilitate the formation of the transition state by decreasing  G ‡ 4

5. Factors influencing an enzymatic reaction 1. Substrate concentration 2. Temperature 3. Inhibitors 4. Activators 5. pH Factors influencing an enzymatic reaction 1. Substrate concentration 2. Temperature 3. Inhibitors 4. Activators 5. pH 5

6. 1. S UBSTRATE C ONCENTRATE At low values of [S], the initial velocity, V i, rises almost linearly with increasing [S]. But as [S] increases, the gains in V i level off (forming a rectangular hyperbola). The asymptote represents the maximum velocity of the reaction, designated V max At low values of [S], the initial velocity, V i, rises almost linearly with increasing [S]. But as [S] increases, the gains in V i level off (forming a rectangular hyperbola). The asymptote represents the maximum velocity of the reaction, designated V max 6

7. The substrate concentration that produces a V i that is one-half of V max is designated the Michaelis-Menten constant, K m is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the K m, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate). 7

8. A LLOSTERIC EFFECTORS Noncovalently bind and regulate the enzyme. The effector may be stimulatory or inhibitory. The substrate and effector usually occupy different specific binding sites. Noncovalently bind and regulate the enzyme. The effector may be stimulatory or inhibitory. The substrate and effector usually occupy different specific binding sites. 8

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11. B ACKGROUND 11 The reaction catalyzed by alkaline Phosphatase  Function R-PO 4 + H 2 O → R-OH + H 3 PO 4 substrate product product  Function R-PO 4 + H 2 O → R-OH + H 3 PO 4 substrate product product

12. E XPERIMENT Facts.. An assay is necessary to study an enzyme The assay is a measurement of a chemical reaction, which might involve measuring the formation of the product (or otherwise the decrease in substrate conc.) Facts.. An assay is necessary to study an enzyme The assay is a measurement of a chemical reaction, which might involve measuring the formation of the product (or otherwise the decrease in substrate conc.) 12

13. R EAGENTS AND INSTRUMENTS 18 labeled plastic tubes Micropipette Spectrophotometer ALP enzyme kit (ready to use) Blood serum 5 N NaOH solution 18 labeled plastic tubes Micropipette Spectrophotometer ALP enzyme kit (ready to use) Blood serum 5 N NaOH solution 13

14. P ROCEDURE Take 18 clean plastic tubes and label them from 1 to 18. Another tube will be used as a blank. This will contain all the reagents except of the enzyme. Make the substrate and buffer concentrations as described in the given table (make sure to keep the total volume of all tubes stable at 1.9 ml). Transfer 100 µl of serum to each tube. Mix substrate and serum solutions and incubate at 37c for 50 seconds. Add 0.5 ml of 5 N NaOH in each tube to stop the reaction. Read the absorbance at 400 nm. Plot reaction rate ( V i ) on Y axis against substrate concentration [ S ] on X axis. Take 18 clean plastic tubes and label them from 1 to 18. Another tube will be used as a blank. This will contain all the reagents except of the enzyme. Make the substrate and buffer concentrations as described in the given table (make sure to keep the total volume of all tubes stable at 1.9 ml). Transfer 100 µl of serum to each tube. Mix substrate and serum solutions and incubate at 37c for 50 seconds. Add 0.5 ml of 5 N NaOH in each tube to stop the reaction. Read the absorbance at 400 nm. Plot reaction rate ( V i ) on Y axis against substrate concentration [ S ] on X axis. 14

15. Tube Buffer μl Substrate μl Enzyme μl 5 N NaOH μl Abs. at 400nm 11001800100500 22001700100500 33001600100500 44001500100500 5 1400100500 66001300100500 77001200100500 88001100100500 99001000100500 101000900100500 111100800100500 121200700100500 131300600100500 141400500100500 151500400100500 161600300100500 171700200100500 181800100 500 15