1. Lab Activity4
IUG, 2015
Dr. Tarek M. Zaida

2. Enzyme Kinetics

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

4. Enzymes decrease activation energy
Biochem Exploring Genes Edward Walker
7/19/2019
Enzymes 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‡

5. Enzyme Kinetics Factors influencing an enzymatic reaction
Substrate concentration
Temperature
Inhibitors
Activators pH

6. 1. Substrate Concentrate
At low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S].
But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola).
The asymptote represents the maximum velocity of the reaction, designated Vmax

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

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

10. Enzyme Kinetics of Alkaline Phosphatase (ALP)

11. Background substrate product product Function
R-PO H2O → R-OH H3PO4
substrate product product
The reaction catalyzed by alkaline Phosphatase

12. Experiment 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.)

13. Reagents and instruments
18 labeled plastic tubes
Micropipette
Spectrophotometer
ALP enzyme kit (ready to use)
Blood serum
5 N NaOH solution

14. Procedure
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 (Vi) on Y axis against substrate concentration [S] on X axis.

15. Abs. at 400nm Tube Substrate μl Buffer Enzyme 5 N NaOH 1 100 1800 5002
200
1700 3
300
1600 4
400
1500 5
1400 6
600
1300 7
700
1200 8
800
1100 9
900
1000 10 11 12 13 14 15 16 17 18