The effect of substrate concentration on catalytic reaction rate and the determination of Mie constant Km and maximum reaction velocity Vmax

Summary

In this experiment, the Km value of sucrase was calculated by using the amount of products (glucose and fructose) formed by sucrase hydrolyzing different concentrations of sucrose with sucrose as the substrate.

Operation method

Dinitrosalicylic acid method

Principle

According to the Michaelis-Menten equation, the Lineweaver-Burk double inverse numerical line equation can be obtained: the intercept on the 1/ V vertical axis is 1/ Vmax , and the intercept on the 1/ [ S ] horizontal axis is -1/ Km . Determination of Km and Vmax, especially Km, is one of the basic contents of enzymology research, Km is a basic characteristic constant of enzyme, which contains the nature of enzyme and substrate binding and dissociation, especially when the same kind of enzyme can act on several different substrates, the Mie constant Km often reflects the strength of the enzyme's affinity with the various substrates, the larger the value of Km, it means that the enzyme and the substrate's affinity is weaker, on the contrary, the larger the value of Km, it means that the enzyme and the substrate's affinity is weaker, on the other hand, the larger the value of Km is, the more the enzyme is, the more the enzyme's affinity is weaker. The larger the Km value, the weaker the affinity between the enzyme and the substrate, on the contrary, the smaller the Km value, the stronger the affinity between the enzyme and the substrate. The double inverse graphical method is the most widely used, and its advantages are: (1) it can accurately determine Km and Vmax; (2) it is easy to see whether the reaction violates Michaelis-Menten kinetics according to whether it deviates from the linearity or not; (3) it can analyze the effects of various inhibitors more easily. The disadvantage of this method is that the experimental points are not uniform, and the error of V hour is very large. It is suggested to use a new Eisenthal straight-line graphing method, that is, to change the Michaelis-Menten equation to: when graphing, intercept each pair of experimental values on the vertical and horizontal axes: V1 ～ [S]1; V2 ～ [S]2; V3 ～ [S]3; ---- connecting the bisecting points to obtain a number of straight lines intersecting at one point, and then we can get the Km and Vmax from this point. The advantages of this graphical method are: (i) it is not used for double inverse calculations; (ii) it is easy to identify those incorrect measurements. The Hanes equation can also be plotted with a slope of 1/Vmax and intercepts of -Km and Km/Vma x : (a) the slope of the line is 1/Vmax and the intercept is -Km and Km/Vma x : (b) the slope of the line is 1/Vmax.

Materials and Instruments

Sucrose
Acetic acid buffer Glucose Fructose Sucrose Dinitrosalicylic acid solution
Spectrophotometer Water bath Test tubes Cling film

Move

1. In this experiment and the next one, the dinitrosalicylic acid method is used to measure the reaction product reducing sugar, in order to grasp the range of this method, a standard curve can be made first. According to the following table for experimental operations.

The first tube was used as a blank control, and the absorbance A520 at 520 nm was measured in the remaining tubes. The A520 value was used to graph the umole number of reducing sugars.
2. Determine the effect of different substrate concentrations on the catalytic rate according to Table 2 below.



In order to make the Km measurement accurate, it is necessary to add sucrose first, pipette accurately, time accurately, add enzyme every 30 seconds or 1 minute, shake the test tube after adding enzyme, and then add enzyme to each tube.After adding the enzyme, the tubes should be shaken and each tube should be reacted accurately for 5 min, then 1.0 ml of dinitrosalicylic acid reagent should be added, and the tubes should be immediately covered with plastic wrap, wrapped with a rubber band, and a small hole should be pierced by a needle, and several tubes should be put into a boiling water bath with a rubber band, and then taken out of the tubes and put them into cold water to cool down quickly after 5 min of boiling. Finally, add 3.0 ml of H2O, shake well, if necessary, a small piece of plastic wrap can be used to cover the mouth of the tube, repeatedly inverted the test tube, mix well. Measurement with a colorimetric cup, the blank control tube solution must be shaken thoroughly to remove air bubbles, A520 value to check the reference cup wall whether there are bubbles, if so, must be poured back to the original test tube, and then shaking to remove residual air bubbles. It is not allowed to suck the reagent by mouth during the experiment. Wash your hands after the experiment.
3. In tubes 9 and 10, NaOH solution is added first to stop the reaction, and then the enzyme is added to ensure that no reducing sugar is produced after the enzyme is added to correct for hydrolysis of the sucrose reagent itself and acid hydrolysis. Correct the A520 values for each tube by drawing a straight line with the data from tubes 9 and 10 to find the corrected data for each of the other tubes, and then calculate [ S ], 1/ [ S ], V, and 1/ V for each tube.
4. Draw a graph of reaction velocity V versus substrate concentration [ S ] (Mie curve) and a graph of the double inverse of 1/ V-1/ [ S ] (do not use the A520 value directly), and calculate the reaction velocity V.Km and Vmax were calculated and compared with the literature values. When recording the experimental results, the units of [ S ] and V should be expressed clearly, and the formula of reaction rate should be listed.

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