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Lab 5-Enzyme

Question 1: How will increasing substrate concentration affect the rate (Voinitial velocity) of an enzymatic reaction if the enzyme concentration is held constant for the different trials? Hypothesis: An increase in substrate concentration with increase the rate until the enzyme is saturated. Argument in support of the hypothesis: An enzyme is a macromolecule that serves as a catalyst, a chemical agent that increases the rate of reaction without being consumed by the reaction. Most enzymes are proteins (Campbell “Biology” 8th ed. p. G-12). In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. The catalytic cycle of an enzyme occurs in three steps. The enzyme and the substrate bind to form an enzymesubstrate complex. While bound tot he enzyme, the substrate is converted to a product resulting in an enzyme-product complex. The product is then released and the enzyme is free to bind another substrate molecule(Bio 05LA Lab Manual lab 5, p.1).The part of the active site that binds the substrate is the enzyme-substrate complex, which is where the substrates are held in the active site by weak interactions, such as hydrogen bonds and ionic bonds. The catalytic cycle of an enzyme is as follows, first the substrate enters the active site allowing the enzyme to change shapes, second, the substrate then held in active site by weak bonds, third, at this point the active site can either lower activation energy and speed up the reaction, fourth, it then converts the substrates into products, fifth, the products are then released, finally the active site is restored and now available for two new substrates (Campbell “Biology” 8th ed. p. 155). The step of the cycle where rate limiting occurs is step four, where the substrates are converted into products. The substrate concentration could influence the rate limiting step by the more substrate molecules that are available, the more frequently they access the active sites of the enzyme molecules. Nonetheless, there is a limit to how fast the reaction can be pushed by adding more substrate to a fixed concentration of enzyme. During some point, the concentration of substrate will be high enough that all enzyme molecules have their active sites engaged. As the product exists an active site, another substrate molecule enters. At this substrate concentration the enzyme is said to be saturated and the rate of reaction is determined by the speed at which the active site converts the substrate into a product. Once it is saturated the only way to increase the rate of product formation is to add more enzymes. Cells increase rate of reaction by producing more enzyme molecules. (Campbell “Biology” 8th ed. p. 155). Experimental strategy: I am going to test different substrate concentrations. We will obtain various tubes and add 4mL of different substrate concentrations to the tubes. It is necessary to re-zero the spectrophotometer. We will then begin with the adding of the enzyme topped with parafilm, mix gently by inverting tube three times, immediately put into spectrophotographer. Immediately take absorbance readings and again once

We will plot absorbance vs. Lastly. which means that the graph plot will increase in absorbance and increase in time/rate for each trial. plot the absorbance versus time/rate on the graph. time for each experimental trial. . If my hypothesis is correct. an increase in substrate concentration with increase the rate until the enzyme is saturated. The plot with the steepest slope will be the one that has lowest time/rate and lowest absorbance. Prediction: For each substrate concentration we will end up with one point/plot on the graph.enzyme has been added.