Kinetic Parameters of Catalase
Aya Essam El Garhy
Ola El Rakhawy
Students in Biochemistry Lab
at German University in Cairo
Abstract
The activity of catalase enzyme towards hydrogen peroxide is tested by measuring the amount of H2O2 consumed by the enzyme catalase (by determining the decrease in H2O2 concentration) within a time interval of 5 minutes. Then ending the reaction by the addition of H2SO4 (by changing the PH of the medium). The reaction is carried out with different H2O2 concentrations and a blank is done for each concentration then the amount of remaining H2O2 is determined by titration versus KMnO4. The titration is done for both reaction mixture and blank and the difference between the two values is equal to the amount of H2O2 that was decomposed by the enzyme catalase . The results are then plotted and analyzed according to Michaelis-Menten and Lineweaver- Burk and the values of Km and Vmax are found from the graphs.
Introduction:
Catalase (EC 1.11.1.6) is one of the most potent catalysts now. The reactions it catalyses are crucial to life (1). Catalase catalyzes conversion of Hydrogen peroxide, a powerful and potentially harmful oxidizing agent, to water and molecular oxygen (1). The overall reaction of the enzyme is:
2 H2O2 -- 2 H20 + O2
Catalaze also uses hydrogen peroxide to oxidize toxins including Phenols, Formic acid, Formaldehyde, and Alcohols (2).
The protein has a dumpbell-shaped structure consisting of a tetramer of four identical subunits (220,000 to 350,000 kD) (2). Each monomer contains an iron cofactor bound in a prosthetic heme group (2). All catalases have the same overall structure, but some differ in the number and individuality of domains (1). Some catalase monomers also contain one tightly bound NADP in each subunit (3). This NADP may serve to protect the enzyme from oxidation by its H2O2 substrate (3).
Catalase was one of the first enzymes to be purified to homogeneity, and has been studied intensely...