Paranitrophenol Phosphate Reaction

Enzymes are found in all living cells as part of their protein constitution, which means that they are made of chains of amino acids bound together. These molecules provide energy for the organisms by catalyzing various biochemical reactions. Most of these chemical reactions would not take place in the conditions available if the enzymes were not a present. Therefore, we can say that being involved as catalysts is the main and most important role of an enzyme in any organism. Furthermore, many reactions that are thermodynamically favored do not occur quickly because there is not enough energy necessary to start the reaction (1). This energy is called the activation energy and the role of the enzyme is to provide energy input to reach that threshold
and launch the reaction; otherwise the reaction would not take place even if it is exergonic and highly thermodynamically favorable. That being said, the energy provided by the enzymes lowers the activation energy required for the reaction to happen, and as such they increase the rate at which the reaction occurs. In general, the main idea is that enzymes increase the rate of a reaction by lowering the activation energy (1, 2).
Enzymes have different properties that are worthy to take note of and to consider when dealing with different reactions. The first aspect is denaturation. Enzymes, as we mentioned before, are proteins. Thus, it is necessary that they maintain their three dimensional structure so that their function is preserved and not altered. We also know that proteins structures are generally changed when there is a change in pH, temperature or ionic strength which disrupts the different bonds and the general structure of the proteins, leading to a loss or change in function. That being said, it is necessary to always keep in mind the optimal conditions of an enzyme for it to properly function (3, 4, 5). A second property of enzymes is specificity. Enzymes catalyze specific reactions by binding to the reactant, also called the substrate, in the active site, which leads to the formation of the products. The active site is specific for the substrates so that almost no other substances will bind there, and subsequently react. In a simplistic manner, this is similar to the relationship of a lock and key. Thus each enzyme will catalyze only one specific reaction (3).
The third and last most important property of enzymes is regulation. In an organism, a constantly active enzyme is not always good, therefore cells have the ability to upregulate or downregulate the activation of the enzymes through different methods including allosteric regulation and covalent modification (3).
Enzyme kinetics is the study of reaction rates catalyzed by enzymes and the factors that have an effect on that catalysis and the properties of the enzyme.
In this experiment, a reaction is considered where a colorless Paranitrophenol Phosphate (PNPP) is converted to the yellow paranitrophenol (PNP) through the action of an enzyme called alkaline phosphatase. This enzyme normally acts on molecules that have a phosphate group by cleaving it. The main purpose of the experiment is to study the kinetics of alkaline phosphatase when acting on a conversion of PNPP to PNP by measuring the activity of the enzyme in terms of the amount of the product made. Since PNP has a yellow color, we make use of the spectrophotometry technique for absorbance at 420 nm to keep track of how much product is there (6). The experiment is performed in three main segments: in the first one, a standard curve of PNP absorbance in terms of nmoles of PNP is generated so that all absorbance values obtained later on in the experiment can be converted to a concentration value. The second part of the experiment is an enzyme optimization assay so as to determine the best enzyme concentration to be used for the rest of the assays. In the third segment, once the enzyme concentration is fixed, the substrate concentration is then varied as so to obtain the Michaelis-Menten and Lineweaver-Burk data by determining the velocity in terms of substrate concentration, and eventually determining values for Vmax, Km and the specific activity as an ultimate purpose of this experiment. Vmax is the maximal velocity of the reaction that is reached when all the enzymes are being bound to the substrate and no more are available. Km is a parameter that represents the substrate concentration that corresponds to half of the maximal velocity Vmax. Km is also a good measurement of the affinity of the enzyme where the higher its value, the lower is the affinity (3, 4,
7).
Alkaline phosphatase is an enzyme that dephosphorylates different types of molecules including proteins and nucleic acids. The specific type of enzyme used in this experiment is bovine alkaline phosphatase which is a dimeric glycoprotein that is stable in the pH range of 7.5-9.5. The pH optimum is known to change depending on the substrate and the substrate concentration among many other factors. In the industry field, alkaline phosphatase is used in the determination of how effective the pasteurization of dairy products is. This is based on the fact that alkaline phosphatase denatures at temperatures around 72 degrees Celsius while most milk-born pathogens are known to be destroyed at less extreme temperatures. Thus, the measurement of alkaline phosphatase activity can determine the effectiveness of the pasteurization. It is also important to note that the reaction described above (conversion of PNPP to PNP) is one method to perform this measurement in this context (8).
Overall, the purpose of this experiment is to study the kinetics of alkaline phosphatase through the reaction involving the conversion of PNPP to PNP. This is based on the measurement of the amount of PNP produced that reflects the activity of the alkaline phosphatase base on reading of spectrophotometry. The generation of a Michaelis-Menten plot as well as a Lineweaver-Burk graph allows the determination of all the different kinetic parameters including Vmax, Km and the specificity (6).