The purpose of this experiment is to measure the rates of reaction of the enzyme Alkaline Phosphatase with the substrate p-nitrophenol phosphate under varying conditions. The concentration of both substrate and enzyme was diluted and the inhibitor vanadate was utilized to determine whether or not the reaction is substrate or enzyme-dependent and to see what type of inhibition vanadate was involved.
A class of proteins called enzymes catalyzes almost every chemical reaction in a cell. Enzymes increase the rates of reaction for those reactions, which are already energetically favourable, by lowering the activation energy. Enzymatic reactions differ from other chemical reactions, by having a higher reaction rate, greater specificity, and high capacity for regulation. Quite often, the rate of an enzymatically catalyzed reaction is 106-1010 times that of an uncatalyzed reaction under similar conditions. Enzymes are most effective under the optimal conditions of a cell, in which the cells aqueous environment is 37° C, and has a pH between 6.5-7.5.
Enzyme kinetics, the rate of reaction, and how this rate is influenced by different factors are directly correlated to the path followed by the reaction. For example, the enzyme-substrate reaction rate can be affected when there is a competitive inhibitor is involved. In the reaction, the competitive inhibitor competes with the substrate for the enzyme’s active site. This results in a lower reaction rate of the enzyme-substrate. On the other hand, noncompetitive inhibitors do not compete with the substrate for the active site and will not affect the affinity of the enzyme for its substrate, however, it will affect the maximum velocity of the reaction.
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The catalytic action of an enzyme on a given substrate can be described by two parameters: Km (the Michaelis constant), which measures the affinity of an enzyme for its substrate, and Vmax, which measures the maximal velocity of the reaction at saturating substrate concentration. From the Michaelis-Menton complex:
E + S « ES « E + P
Where E is the enzyme, S is the substrate, and P is the product. The rate of product formation V can be determined by the equation below.
V= Vmax [S]/[S] + Km
From this equation, we can predict that when the V is independent of [S] the reaction would be zero-order, whereas when V is dependent on [S], the reaction is first order. Km and Vmax can be calculated from the Lineweaver-Burke plot. This is done by taking the double reciprocal of the Michaelis-Menten equation and is illustrated graphically.
Another way to interoperate the Vmax and Km is by Eadie-Hofstee
With this knowledge and the utility of the spectrophotometer, we were able to determine what kind of inhibitor vanadate was and whether or not the reaction was substrate or enzyme dependant.
There are other enzymes besides Alkaline Phosphatase that demonstrate Michaelis-Menten kinetics such as Amylases, Glucose Isomerase, Invertase, Lactase, Glucose Oxidase-Catalase, Polyphenol Oxidase and Pectinases. Alkaline Phosphatase is normally present in high concentrations in growing bone and in bile. It is essential for the deposition of minerals in the bones and teeth. Alkaline phosphatase deficiency is a hereditary trait called hypophosphatasia, which results in bone deformities
The methods used for this experiment were taken from the Biology 366 Lab Manual, written by Segal.
In section A the initial dilution of 1/6 was changed to 1/5 in order to get a preferred reaction rate.
There were three major sections in this lab; the first was called Demonstration of the enzyme assay. Here a proper dilution of Alkaline Phosphatase, stock enzyme, was taken and because of this enzymes reactive properties which produce a yellow (405 nm) colour one was able to quantify the amount and the rate of the reaction by a spectrophotometer. This portion is done to get the proper activity of the enzyme.
The next portion of the lab is titled Effect of varying substrate concentration (enzyme concentration kept constant) with and without (vanadate). This portion was done to determine if vanadate is a competitive or non-competitive inhibitor. Here different concentrations of the substrate were made and there were two runs, one with the Vanadate and one without. Again the abortions were taken and visually graphed in three types of graphs, Michaelis-Menten, Lineweaver-Burke, and Eadie-Hosftee plots, to determine the type of inhibition that was taking place.
The last Portion was called the Effect of varying enzyme concentration (Substrate concentration kept constant). Here the different dilutions of the enzyme were recorded to establish if the amount of enzyme played a role in the rate of the reaction.
Altering the reaction rate by varying the [S] without changing the [E], in the presence and in the absence of the inhibitor vanadate. From the graph, we can see that the inhibitor vanadate is a noncompetitive inhibitor. The curve of the inhibitor is not similar to that of the uninhibited in that, both have the different Vmax; the results indicated that as the [E] increased, the interaction between enzyme and substrate increased, the reaction rate also increased.
This result follows the saying more is better. As more enzymes were added the rate of reaction increased. Also, the concentration of the substrate should be higher than the enzyme because if it was not the graph should show a levelling off. Furthermore, the initial reaction rate represents the Vmax because there are fewer enzymes in the substrate therefore each enzyme has more opportunity to bind to the substrate. All these equations have their benefits and mostly they are all derived from the Michaelis-Menten plot, but personally, I preferred the Lineweaver-Burkes plot, it seemed that the Vm and Km were easier to derive.
The results generated from this experiment indicate that the reaction rate directly depends on the concentration of the enzyme. The reaction rate increases with the increase in enzyme concentration. From the Michaelis-Menten graph, we can determine that the reaction rate is exponentially related to the substrate concentration, the Lineweaver-Burke shows the effect of an inhibitor, vanadate, as a non-competitive inhibitor. Also in my lab group, there was an error in making the proper dilution for the second section which explains the not so accurate picture in the Michaelis-Menten graph.
Lodish, Molecular Cell Biology, 4th ad. W.H. Freeman and Co. NY.2000
Enzyme Concentration From: http://www.worthington-bi
ochem.com/introBiochem/enzymeConc.html . October 30, 2001
Biology 366 lab Manual, Segal.
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