St6gal-1 Lab Report

Background and Introduction: Enzymes are proteins that process substrates, which is the chemical molecule that enzymes work on to make products. Enzyme purpose is to increase the rate of activity and speed up chemical reaction in a form of biological catalysts. The enzymes specialize in lowering the activation energy to start the process. Enzymes are very specific in their process, each substrate is designed to fit with a specific substrate and the enzyme and substrate link at the active site. The binding of a substrate to the active site of an enzyme is a very specific interaction. Active sites are clefts or grooves on the surface of an enzyme, usually composed of amino acids from different parts of the polypeptide chain that are brought together in the tertiary structure of the folded protein. Substrates initially bind to the active site by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Once a substrate is bound to the active site of an enzyme, multiple mechanisms can accelerate its conversion to the product of the reaction. But sometimes, these enzymes fail or succeed to increase the rate of action because of various factors that limit the action. These factors can be known as temperature, acidity levels (pH), enzyme and/or substrate concentration, etc. In this experiment, it will be tested how much of an effect

Since most of the known biological catalysts are proteins two criteria are generally used for establishing the existence of enzymes. The first is that the rate of a reaction in the presence of an enzyme is greater than the rate in its absence. Because the uncatalyzed rates of most biologically important reactions are effectively zero, the mere

Enzymes are very large globular proteins with three dimensional shapes which is vital for enzyme activity as natural catalyst in chemical reactions within the living organisms (7).

J. Moldovan & B. Nilson, (2010), Lab 4 – Enzyme Kinetics, UBCO BIOL/BIOC 393, UBC Vista accessed Monday, November 8th, 2010.

Describe an experiment (assay) that could be used to determine the kinetic parameters of your enzyme.

The enzyme can change shape when bound by the substrate, which is triggered by the strong magnetic forces during binding. This change in shape aligns the two substrates in the positions in which they are connected by the hook-and-loop fastener (Figure 2). This simulation shows important features of the induced-fit model: sub- strate binding induces a change in the shape of the enzyme, which brings about the catalysis of the substrates into the product. This simulation helps tackle the misconceptions that the substrates bind with the enzyme in a lock-and-key manner and that it is the sub- strate, rather than the enzyme, that undergoes induced fit (College Board,

Graph 6a (Substrate) – Represents a xy scatterplot with linear regression, which show the change in product concentration over the change in time at different substrate The data in the graph also gives the value of V0 at corresponding substrate concentration. Graph 6b (Substrate) – Represents a xy scatterplot depicting velocity of enzyme-catalyzed at multiple substrate concentrations. To find the concentration of the different absorbance in this experiment, a modified version of the Beer’s Law equation was used (C=A/k). The k which represents the slope in the equation was determine by using the date from Table 1 and points plotted in Graph 1. k=6.8339 the A in the equation is the measured absorbance which was determine by using a spectrophotometer.

Enzymes are biological catalysts that are responsible for the biochemical reactions inside living cells. They are made up of proteins that are synthesised by only living cells and speeds up the rate of reaction by up to a million times (Greenwood, 2011). Temperature, pH levels, substrate and enzyme concentration and inhibitors affects the enzyme action (Rsc.org, 2016). Two theories are used to describe the mechanisms of enzyme activity; the lock and key theory and the induced fit mechanism.

References: "Biomolecules:Enzymes." Competitive Inhibition. Chempages Netorials, 2006. Web. .<http://www.chem.wisc.edu/deptfiles/genchem/netorial/modules/biomolecules/modules/enzymes/enzyme5.htm>.

When unlocking a door, only a certain shape and size of a key will fit into the lock. This is similar with and enzyme and a substrate. Only a specific shape and size substrate will fit into the enzyme’s active site (figure 1B). The induced fit model explains how an enzyme’s active site many change after the enzyme and the substrate are combined (figure

The lock and key model has been one theory that has been proposed to show how enzymes work. This model shows how a substrate is binded to the enzyme’s active site. This model further shows how specific and accurate the substrate has to be in terms of its complementary shape and size in order to fit in into the enzyme’s active site. As a result of this structure, only certain amount of compounds will be able to fit successfully. When the substrate and active site bind to one another, a chemical reaction takes place that eventually will release the products that have been formed, an enzyme-substrate complex will be formed once the interaction of the substrate and active site of the enzyme happen. An enzyme-product complex can be made which can

In this report we tried to model the rate law of a reaction with an enzyme catalyst using the Michaelis-Menten model, which predicts the rate law to be:

Enzymes are natural catalysts that work from the ability to increase the rate of reaction by decreasing the activation energy of a reaction. (Blanco, Blanco 2017) An enzyme can do this 10^8- to 10^10 fold, sometimes even 10^15 fold. (Malacinsk, Freifelder 1998) The substrate will momentarily bind with the enzyme making the enzyme-substrate complex, of which the shape of the substrate is complimentary to the shape of the active site on the enzyme it is binding with. There are two main theories as to how an enzymes and substrates interact, the lock-and-key model and induced fit theory. The lock-and-key model suggests that the enzyme has a specific shape that fits the substrate and only that substrate. The induced fit theory says the active site and substrate are able to change shape or distort for the reaction to take place with (Cooper,

T4 lysozymes (T4L) were added to 2AR to stabilize 2AR- Gs. A previous paper referenced by the Kobilka group (3) used this approach to put T4L between TM 5 and TM 6. In it this Science paper, they expressed concern about T4L addition modifying WT 2AR. What they found were minimal differences in the modified 2AR compared to WT (normal antagonist binding and slightly elevated agonist binding) (3). I would have liked to have seen a similar functional comparison done for the WT-2AR compared to the T4L modified 2AR for this paper as well for completeness to ensure T4L addition does not change function. Another problem was the variability in the position of GsAH. A nanobody (Nb35) was bound to the complex to help reduce this variability. As with T4L addition, I would have liked to have seen additional data included to show that the Nb35 addition does not significantly alter the function of the protein complex. While Figure 2c) in the paper shows the structure of the structure of the 2AR- Gs complex without the aids, I would have liked to have seen some functional tests performed on the complex, both with the aids for crystallization and without the aids for crystallization.

Objective 3: Rational engineering of dynamic crystals to fit a wide variety of enzymes and substrates.