The majority of chemical reactions that take place within living cells require highly specific protein catalysts called enzymes. Enzyme action can be altered through changing conditions. Experimental procedure is used to examine important principles of enzymes and enzyme assays, and to investigate enzyme kinetics. Results show enzyme action to be optimal at a pH around 9, and at higher temperatures in conditions lacking inhibitor molecules. The enzyme activity rates can be measured using a spectrophotometer, which gave absorbance readings that were then converted into rate figures. Results show that higher absorbance readings give a higher enzyme reaction rate. Higher reaction rates demonstrate optimal conditions for enzyme activity.
Enzymes function as a catalyst. Many biological processes require enzymes for the reaction to take place in living cells. Enzymes are large proteins with specific 3-dimensional structures that enable them to bind to specific substrate. As they are catalysts, this means they can speed up chemical reactions through lowering the activation energy. The rate at which a reaction takes place is impacted by several factors such as enzyme concentration, temperature, pH and the presence of activator or inhibitors. Enzymes are charged molecules due to the presence of amino acids, their charge is dependent on the pH of surrounding solution. Enzyme assays using alkaline phosphatase demonstrate the typical characteristics of most enzymes. Alkaline phosphatase operates optimally in alkaline pH conditions, hence its name, and acts widely on a range of organic monophosphate esters, hydrolysing them into an organic alcohol and inorganic phosphate. The amount of enzyme activity can be demonstrated by measuring the rate of appearance of product. The substrate p-nitrophenyl phosphate (pNPP) is colourless when in pH conditions of 7. The product of the reaction, p-nitrophenol is yellow in alkaline conditions. Colorimetry detects the concentration of solution identifying the amount of product there is. The reaction takes place at different rates dependent upon the surrounding environment. When exposed to extreme temperature or pH it causes the non-covalent bonds within the structure to break, changing the 3-dimensional shape of the enzyme causing enzyme denaturation. The enzyme can no longer function as the active site is no longer complimentary to the substrate. Thus, there are optimal conditions for enzymes to work best at.
The aims of an enzyme activity experiment are to examine the important principles of enzymes and enzymes assays. Additionally, to introduce the study of enzyme kinetics through measuring enzyme activity at changing pH, temperature, the addition of an enzyme inhibitor, and determining the Michaelis-menten constant and the order of reaction.
The experimental procedure will consist of incubation of alkaline phosphatase with the substrate p-nitrophenyl phosphate at 37°C and pH 9.5, leading to the production of the product p-nitrophenyl. The effects of changing temperature, pH, and presence of inhibitor will be tested and a spectrophotometer will be used to measure the absorbance of sample solutions at 410 nm to measure the hydrolysis of pNPP. Measuring the hydrolysis requires the addition of NaOH to make the solution alkaline and to denature the enzyme after reaction completion. The reaction velocity and order of reaction can be calculated using the concentrations of product.