V_max and K_m are the characteristic properties of an enzyme. As is clear from equation 12.1, K_m can be defined as the substrate concentration at which initial reaction rate, .

The response of enzymes to the concentrations of substrates and products plays important role in the reaction control. This behavior of enzymes to the substrate/product concentration is studied under enzyme kinetics and is used to determine the important enzyme parameters such as K_m and V_max. We have chosen to study the kinetics of the enzyme alkaline phosphatase. The enzyme catalyses the hydrolysis of a phosphoester bond, producing inorganic phosphate (P_i) and an alcohol. We have chosen p-nitrophenylphosphate as the substrate for the hydrolysis reaction. Para-nitrophenylphosphate is a colourless compound; the enzyme, alkaline phosphatase hydrolyses the phosphoester bond to produce the coloured product, p-nitrophenol which can be detected colorimetrically (Figure 12.2).

Figure 12.2: Hydrolysis of p -nitrophenylphosphate into p -nitrophenol and phosphate

Materials:

Equipments:

1. UV/Visible spectrophotometer
2. Weighing balance

Reagents:

1. 100 mM Tris-HCl buffer, pH 8.0
2. Para-nitrophenol (PNP)
3. Para-nitrophenylphosphate (PNPP)
4. Alkaline phosphatase from E. coli

Glassware, plasticware, etc.:

1. 1.5 ml microfuge tubes
2. Pipettes
3. Pipette tips
4. A pair of matched glass or quartz cuvettes (volume: 3 ml )

Procedure:

Standard curve of PNP

1. Switch ON the spectrophotometer and allow it 30 min warm up.
   
   
2. Meanwhile, prepare 0.1 mM PNP solution in 100 mM Tris-HCl buffer, pH 8.0
   
   
3. Take 11 microfuge tubes and label them from 1 – 11.
   
   
4. Prepare the PNP dilutions as shown in table 12.1.