Enzymes are proteins that function as biological catalyst. Enzymes increase the rate of a reaction without being consumed themselves by the reaction. Enzymes can speed up a chemical reaction by lowering the activation (EA ) barrier. (1) Enzymes speed up a chemical reaction and lower the activation barrier of a chemical reaction by binding to the reactant or substrate using their active sites. All enzymes has an active site, which is a pocket on the surface of the enzyme that fit into its substrate. (1) Enzymes are specific because of their active sites that can only fit specific substrate molecule. (1) There are four steps in an enzyme-substrate reaction:
Step (1) begins when enzyme with an empty active site flowing freely around looking for its substrate.
Step (2) starts when the active site of an enzyme E bind to the substrate S, attaching it by weak bonds. The active site changes shape slightly so that it embraces the binding of the substrate more firmly. The substrate binds to the enzyme with induced fit forming an enzyme-substrate complex (ES).
Step (3) happen when the enzyme-substrate complex (ES) react with water, and the substrate s is hydrolyzed into its product. (1) The enzyme and product are still chemically bound (EP)
Step (4) happens when the enzyme releases the products and is unchanged from the reaction. The active site of the enzyme is now available for another substrate molecule, beginning another round of the cycle. (1)
The overall activity of an enzymatic reaction is summarized as follows:
E + S --- ES --- EP ----- E + P
The enzyme most frequently study is bovine alkaline phosphatase. Alkaline phosphatase hydrolyze the artificial substrate p-nitrophenylphosphate (pNPP) giving us a way to make quantitative measurements of its activity. pNPP is a chromogenic substrate for alkaline phosphatase. pNPP is a colorless substrate, which becomes yellow when de-phosphorylated by alkaline phosphatase to form the product p-nitrophenol (p-NP).
The reaction of the enzyme alkaline phosphatase
p-NPP ( colorless) + H2O ----------------? p-NP ( yellow) + inorganic phosphate
The rate of color change is directly proportional to the rate of conversion of substrate to product. A device called a spectrophotometer is used to measure this color change as a function of time to determine the rate at which the colored product is made
Experiment #1
Influence of changes in substrate concentration upon reaction rate
Different substrate concentration:
Full strength ( 600 uM)
Half strength ( 300 uM)
No substrate ( 0 uM)
Prepare a sample tube with 4.0 ml of full strength substrate.
Prepare another tube with 4.0 ml half strength substrate.
Prepare a tube with no sample (blank).
Add 1 ml of enzyme alkaline phosphatase. Mix well by inverting the tube three times. Begin timing every interval. Insert into the spectrophotometer and get reading.
Record the data:
Time (sec) Full strength (pH 10) Half strength( pH 10) Zero strength(pH 10)
30 101 65 0
60 142 75 0
90 172 92 0
120 205 107 0
150 240 135 0
180 288 186 0
210 305 201 0
240 360 253 0
270 410 300 0
300 520 350 0
1) What is an enzyme?
2) What are the steps in an enzyme-substrate reaction?
3) How will decreasing the concentration of substrate affect the reaction rate?
Experiment #2 The influence of pretreating the enzyme with a range of temperatures
The effect of different temperature pretreatments of an enzyme on its reaction rate will be examined here. The temperatures used is 40 and 80 ° C.
1. Prepare two water baths. One water bath should be heated to 80°C by placing it on a pre-warmed hot plate. The other water bath should be heated to 40°C by placing it on a pre-warmed hot plate.
2. Label each tube with either 40 °C or 80°C and place 1.0 ml of full strength alkaline phosphatase enzyme into each of the tubes.
3. Place the tubes in the appropriate water bath for 5 min.
4. After 5 min, chill the heat-treated enzyme by pushing the tubes into ice for 5 min.
5. Remove tubes from ice and let them warmed to room temperature.
6. For the 40C tube, add 4.0 ml full strength substrate into the tube, mix, and take readings.
7. For the 80C tube, add 4.0 ml full strength substrate into the tube, mix, and take readings.
Time (se) 40°C 80°C
First 42 21
30 45 22
60 51 25
90 62 29
120 74 34
150 79 38
180 82 42
210 86 48
240 90 52
270 94 59
300 98 63
4) How will pre-treatment of the enzyme with high temperature affect the reaction rate?
Experiment #3 The influence of altering pH on the reaction rate
The effect of a change in pH on reaction rate will also be examined. The pH's to be used will be 6.5.
1. Place 4.0 ml of pH 6.5 substrate in a clean tube.
2. Add 1.0 ml of pH 6.5 enzyme to the tube containing the substrate and take absorbance reading after adding the enzyme.
3. Compare this to pH 10.
Time (sec) pH 10 pH 6.5
First 98 42
30 101 51
60 142 56
90 172 62
120 205 69
150 240 72
180 288 82
210 305 89
240 360 95
270 410 98
300 520 103
5) How will lowering the pH ( making the experimental conditions more acidic) affect reaction rate?