Varying Effects of Enzyme Concentration on Reaction Rates of Malate Dehydrogenase

9 September 2016

There are two different ways in which a substrate can bind to an active site. The first way is through the lock and key model. Certain substrates can bind only to certain active sites in the lock and key model. This is called the lock and key model because the active site is like the lock and the key is the substrate. Only certain keys can open certain locks. This accounts for the specificity; not all substrates can fit into all active sites. Another way in which substrates bind to active sites is called the induced fit model.

This is where an active site changes its form to accommodate for certain substrates. There are several different biological factors that effect enzyme activity. Some of them include temperature, pH, and enzyme concentration. This experiment will be testing how much of an effect different concentrations of enzyme solutions have on reaction rates. There are many different kinds of enzymes. A group of people have formed the Enzyme Commission for organizing and identifying enzymes. They set up a system where each enzyme has a four number name.

Varying Effects of Enzyme Concentration on Reaction Rates of Malate Dehydrogenase Essay Example

The enzyme for this reaction is malate dehydrogenase (MDH). Its enzyme commission number is 1. 1. 1. 37. The first number is in reference to its class. In the case of MDH, it is a oxidoreductase meaning that it carries out oxidation-reduction reactions. The next two numbers refer to the subclass and sub-subclass. The last number is the specific serial number for that enzyme. As mentioned before, MDH carries out an oxidation-reduction reaction where oxaloacetic acid (OAA) is and NADH are oxidized to produce malic acid and NAD+.

The OAA and NADH (reduced form) are the substrates in this reaction and the malic acid and NAD+ are the products that arise from this oxidation-reduction reaction. This specific reaction is very important because it occurs in the Krebs cycle. The Krebs Cycle is what is a series of reactions that take place in the mitochondria to generate ATP which is an energy source for cells. Without this reaction, there would be no life on earth so it is vital to our existence. MDH is obtained from bovine hearts and has been thoroughly researched.

This reaction with MDH has a specific characteristic that enables researches to study its specific reaction rate. NADH absorbs light at 340nm and at the moment when MDH catalyzes the oxidation of NADH, the absorbance decreases. For this reason, concentrations of the enzyme will be tested to see how it affects this important reaction. MATERIALS AND METHODS MDH Assay The following procedures were taken from Boernke et al (YEAR). An assay was run with optimal conditions for MDH (7. 51pH, 25°C, 1x conc. ) to use as a comparison for the results. This was done by mixing 10? L of MDH solution with 1. mL of phosphate buffer, 10? L of OAA, and 10? L of NADH in a cuvette. The cuvette was then mixed by placing a square of parafilm over the top and inverting a few times. This cuvette was immediately placed in a Barnstead/Turner Spectrophotometer model sp-830 to obtain a starting absorbance at 340nm. The absorbance was taken in the spectrophotometer every ten seconds after that for two minutes and recorded. In between each reading, the spectrophotometer was calibrated by taking the absorbance of a ‘blank. ’ The blank contained everything mentioned above except for the 10?

L of MDH. The readings were then used to make a chart where time was plotted against absorbance. By calculating the slope of this chart (? y/? x • 60/1min), the absorbance per minute was achieved, hence, finding out how fast the reaction occurs in nature. Also by dividing the slope by 6. 22, the units of enzyme activity was calculated. Effects of Varying Enzyme Concentration The next part of this experiment was to test the rate of absorbance using different enzyme concentrations. 1. 0mL of phosphate buffer, 10? L of OAA and 10? L of NADH were added 3 different cuvettes.

In the first of those cuvettes 10? L of . 25x MDH dilution was added and run through the same experiment as the MDH assay (mixing and taking the absorbance every ten seconds for two minutes with ‘blanks’ in between). In the second cuvette, 10? L of . 5x MDH dilution was used and in the third cuvette the stock solution of 10? L of 1x dilution of MDH was used. After logging all of the absorbance values of each of the three test tubes, charts were made by plotting absorbance verse time and slopes and units of enzyme activity were calculated from that chart.

Then another chart was made comparing the units of activity with the different enzyme dilutions. RESULTS The results of this experiment found that the stock concentration of 1x showed the most units of activity as Figure 2 suggests. Figure 2 is specifically comparing the units of activity of each of the three concentrations and their corresponding units of activity. The 1x concentration had . 1187 units of activity while the . 5x had . 0816 units and the . 25x had . 0384 units of activity. As seen in Figure 1, the line for the 1x concentration started at a much lower absorbance reading.

It also had the steepest slope showing the reaction occurred more rapidly than that of the . 5x and . 25x. DISCUSSION A reason for why the 1x concentration rose above the others lies in the aspect of evolution and natural selection. As mentioned in the introduction, MDH comes from a cow heart and occurs in nature so it would make sense the over time nature ‘selected’ the 1x concentration of MDH to work the best because when it actually happens, the concentration is not diluted to half for one-fourth of the original solution. Also it could be due to error that the absorbance of the 1x concentration started at a lower value.

Maybe the reaction happened so fast that the reaction occurred before the cuvette was placed in the spectrophotometer. It could have been that it was mixed more thoroughly as well. It would be really interesting another study followed up on this and figured out what exact concentration works the best. They could use very similar concentrations like . 99x 1x and 1. 01x. FIGURES LITERATURE CITED Becker, W, Kleinsmith, L, & Hardin, J (2006). The World of the Cell. San Francisco: Bemjamin Cummings. (Becker, Kleinsmith, and Hardin, 2006)

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