Salicylate (aspirin) inhibits the catalytic action of glutamate dehydrogenase. You measure the activity of this enzyme in the absence and presence of the inhibitor. The data is presented in the table below. Substrate concentration (mM) Micrograms of product per minute, no inhibitor Micrograms of product per minute, 40 mM salicylate 0 0 0 0.5 0.11 0.04 1.5 0.23 0.09 2 0.27 0.1 3 0.32 0.12 4 0.36 0.13 8 0.45 0.16 16 0.5 0.18 In Microsoft Excel, generate the Michaelis-Menten graphs (i.e., the rate versus substrate concentration plots) for each of the inhibitor-free and the inhibitor-containing reactions. Without doing any calculation, and simply by visually observing the plots in Excel, estimate what the Vmax and the Km for this enzymatic reaction in the presence and the absence of the inhibitor. For Vmax, express your answer to one decimal place while using the appropriate rounding. For Km, express your answer to the nearest integer. Enter your answers in the table below. Without inhibitor With inhibitor Km (mM) Vmax (μgmin) A more accurate way to calculate Vmax and Km is to use the double reciprocal plot (i.e., 1/rate versus 1/substrate concentration) because it provides a linear correlation. What is this plot called? It is the - plot. (pay special attention to the spelling, write each name in the appropriate box separated by the hyphen). Using the same excel sheet, plot the same data in the double reciprocal form and fill in the information in the table below. Express all your answers with 4 decimal places. Without Inhibitor With Inhibitor R2 Slope Y-intercept X-intercept Using the X- and Y-intercepts determined above, calculate the Km and Vmax for this enzymatic reaction in the presence and the absence of inhibitor. Express you answers with 2 decimal places. Without inhibitor With inhibitor Km (mM) Vmax (μgmin) Do the estimates determined from the Michaelis-Menten plots agree with the calculations derived from the double reciprocal plots? Answer "yes" if the estimates are within ±10% of the calculated value; otherwise, answer "no". From the data and based on your knowledge of enzyme inhibition, can you determine the type of inhibition (write "competitive", "non-competitive", or "uncompetitive")?
Catalysis and Enzymatic Reactions
Catalysis is the kind of chemical reaction in which the rate (speed) of a reaction is enhanced by the catalyst which is not consumed during the process of reaction and afterward it is removed when the catalyst is not used to make up the impurity in the product. The enzymatic reaction is the reaction that is catalyzed via enzymes.
Lock And Key Model
The lock-and-key model is used to describe the catalytic enzyme activity, based on the interaction between enzyme and substrate. This model considers the lock as an enzyme and the key as a substrate to explain this model. The concept of how a unique distinct key only can have the access to open a particular lock resembles how the specific substrate can only fit into the particular active site of the enzyme. This is significant in understanding the intermolecular interaction between proteins and plays a vital role in drug interaction.
Salicylate (aspirin) inhibits the catalytic action of glutamate dehydrogenase. You measure the activity of this enzyme in the absence and presence of the inhibitor. The data is presented in the table below.
| Substrate concentration (mM) | Micrograms of product per minute, no inhibitor | Micrograms of product per minute, 40 mM salicylate |
| 0 | 0 | 0 |
| 0.5 | 0.11 | 0.04 |
| 1.5 | 0.23 | 0.09 |
| 2 | 0.27 | 0.1 |
| 3 | 0.32 | 0.12 |
| 4 | 0.36 | 0.13 |
| 8 | 0.45 | 0.16 |
| 16 | 0.5 | 0.18 |
In Microsoft Excel, generate the Michaelis-Menten graphs (i.e., the rate versus substrate concentration plots) for each of the inhibitor-free and the inhibitor-containing reactions. Without doing any calculation, and simply by visually observing the plots in Excel, estimate what the Vmax and the Km for this enzymatic reaction in the presence and the absence of the inhibitor. For Vmax, express your answer to one decimal place while using the appropriate rounding. For Km, express your answer to the nearest integer. Enter your answers in the table below.
| Without inhibitor | With inhibitor | |
| Km (mM) | ||
| Vmax (μgmin) |
A more accurate way to calculate Vmax and Km is to use the double reciprocal plot (i.e., 1/rate versus 1/substrate concentration) because it provides a linear correlation. What is this plot called? It is the - plot. (pay special attention to the spelling, write each name in the appropriate box separated by the hyphen).
Using the same excel sheet, plot the same data in the double reciprocal form and fill in the information in the table below. Express all your answers with 4 decimal places.
| Without Inhibitor | With Inhibitor | |
| R2 | ||
| Slope | ||
| Y-intercept | ||
| X-intercept |
Using the X- and Y-intercepts determined above, calculate the Km and Vmax for this enzymatic reaction in the presence and the absence of inhibitor. Express you answers with 2 decimal places.
| Without inhibitor | With inhibitor | |
| Km (mM) | ||
| Vmax (μgmin) |
Do the estimates determined from the Michaelis-Menten plots agree with the calculations derived from the double reciprocal plots? Answer "yes" if the estimates are within ±10% of the calculated value; otherwise, answer "no".
From the data and based on your knowledge of enzyme inhibition, can you determine the type of inhibition (write "competitive", "non-competitive", or "uncompetitive")?
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