Chem 437 Experiment 10-1 and 10-2 PDF

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Lab Report for experiment 10-1 and experiment 10-2.
Experiment 10-1: Determination of Km and Vmax for Alkaline Phosphatase
Experiment 10-2: Product Inhibition of Alkaline Phosphatase...

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Experiment 10-1: Determination of Km and Vmax for Alkaline Phosphatase Experiment 10-2: Product Inhibition of Alkaline Phosphatase

Abstract

The purpose of this experiment is to determine the Km and Vmax of alkaline phosphatase for PNPP substrate as well as the Ki for inorganic phosphate. From our Michaelis-Menten plot, our Km was determined to be .025 and our Vmas was .0014. Our km from our Line Weaver-Burke plot was found to be .027 and vmax was .0016. From our Eadie-Hofstee plot, we found our km to be .021 and our vmax to be .0015. Lastly, we determined that our km was .024 and our vmax was .0015 from our Hanes-Woolf plot. We determined from our Eadie-Hofstee plot that our Kcat was 7.4 and our kcat/km was 352.38 1/mM*sec. Lastly, we performed an inhibition assay to determine the Ki of alkaline phosphatase for Pi and generated a dixon plot which gave a Ki value of 0.0052. Introduction To find Km and Vmax fixed assay was performed using 75 mu/ml of stage 4 enzyme with the addition of increasing PNPP concentration from 0.01 mM to 0.2 mM. The rate of each reaction was determined and then used to generate the following plots: Michalis-Menten, Line Weaver Burke, Eadie-Hofstee, and Hanes-Woolf. The Michaelis-Menten plot shows the hyperbolic relationship between the rate of the reaction and substrate concentration, v= (Vmax[S])/Km. At low substrate concentrations, Km >> [S] and at high substrate concentrations [S] >> Km. At high substrate concentrations v=Vmax and the reaction rate is therefore independent of the substrate concentration. When the velocity of the reaction is half of Vmax Km=[S. This allows us to determine Km graphically. Vmax can also be determined graphically using the Michalis-Menten plot, however, it will be imprecise. Using the inverse of substrate concentration and the inverse of the reaction rate, we can generate a Line-Weaver Burke plot which has a slope that is equal to Km/Vmax, and a y-intercept equal to 1/Vmax. From the slope equation, Km and Vmax can be accurately calculated. The Hanes-Woolf plot is another linear plot like Line-Weaver Burke that can be used to determine Km and Vmax. In this case, S/V vs. [S] is plotted and the slope is equal to 1/Vmax and the y-intercept is equal to Km/Vmax. The Eadie-Hofstee is also a linear plot that is plotted by graphing V vs. V/[PNPP] and has a slope equal to -km and a y-intercept equal to vmax. Kcat is known as the maximum turnover number or catalytic constant that determines how many moles of a substrate are converted by an enzyme to the product per minute when the enzyme is completely saturated under certain environmental conditions. In terms of this experiment, Kcat measures the turnover rate of how much PNPP is converted to PNP per minute when alkaline phosphatase is completely saturated. Competitive inhibition happens when an inhibitor resembles the substrate used by an enzyme. This resemblance causes the inhibitor to bind to the active site of the enzyme which in turn prevents the substrate from binding. Pi acts as a competitive inhibitor of PNPP by binding to the active site of alkaline phosphatase which therefore inhibits the ability for PNPP to bind. To experimentally determine competitive inhibition constants, a series of assays must be performed with at least two different substrate concentrations and with varying inhibitor concentrations. The inverse of velocity of each substrate concentration is plotted against the concentration of the inhibitor which gives a Dixon plot. The intersection of the two lines in the second quadrant gives -ki on the x-axis and 1/vmax on the y-axis.

Materials and Methods Experiment 10-1: Determination of Km and Vmax for Alkaline phosphatase. To begin, we obtained five 13x100 mm test tubes and dispensed different volumes of 0.5 mM PNPP, so the concentration of PNPP in each tube ranges between 30 nmol to 600 nmol. After adding the PNPP, we added 0.2M Tris-HCl to bring the volume to 2.8 ml for each test tube then we mixed. We then blanked the spectrophotometer with the tube that was going to be measured then we added 0.2 ml enzyme to the same tube. We waited 30 minutes and then took the absorbance of each test tube. See table 1 for test tube contents. Contents of Test Tubes for Assay TT #

PNPP (Substart nmol)

Vol. of PNPP needed (mL)

Stage 4 Enzyme (uL)

0.2M Tris

1

600

1.2

200

1.6

2

300

.6

200

2.2

3

150

.3

200

2.5

4

75

.15

200

2.65

5

37.5

.075

200

2.75

Table 1 shows a the contents of each test tube for the assays

Experiment 10-2: Product Inhibition of Alkaline Phosphatase

To begin, we set up 5 test tubes and pipetting 0.3 ml of 0.5 mM PNPP solution to each test tube. After adding the PNPP, we then added enough of the 0.2M Tris-HCl to each tube to make the final volume reach 2.7 ml. Then, we added 0.2 ml of the enzyme solution to each test tube, mix, and allow them to stand at room temperature for about 3 minutes. After 3 minutes, 0.1 ml of 10 M sodium hydroxide was added to each test tube and we mixed quickly. The final volume in these assays should be 3.0 ml. Once mixed, we read the absorbance at 410 nm with a blank that had no enzyme. We did this with every test tube prepared and got the absorbance for each one for 3 minutes. After doing the 5 test tubes we prepare and conduct the same procedure but this time we use 0.9 ml of 0.5 mM PNPP in each test tube.

Results and Discussion When doing our assay, our enzyme activity was very low, so instead of measuring the absorbance every 20 seconds to find our rate we took one absorbance after 30 minutes. We then divided that value by 30

to find the change in absorbance per minute. So we could not make rate plots to compare to the michaelis-Menten plot. Over values for Km and Vmax that were determined from our four different plots were very similar (See table 3). However, all of our Km and Vmax values were very far off from the literature values. The literature value for Vmax is 1.0x10^-8 mM and for the value for Km is .00036. The Eadie-Hofstee plot gave a Km closest to the literature value with a value of .021 and a vmax value of .0015. From these results, we can see that our Km and vmax values are much larger than the literature value, meaning that our alkaline phosphatase had a low affinity for PNPP. This error could have been caused by not isolating alkaline phosphatase correctly and leaving unwanted proteins in the mixture. This error can also be seen when looking at our calculated change in absorbance/minute which is relatively low (See table 2).

Rate Calculated for Tubes 1-5 Tube #

Abs. After 30 Minutes

Calculated Change in Abs/Min

Calculated E (A/.05mm)

Calculate Velocity (mmol/min)

Tube 1

.321

.0107

7.84 mM-1 cm-1

.00136

Tube 2

.269

.0090

7.84 mM-1 cm-1

.00115

Tube 3

.236

.0079

7.84 mM-1 cm-1

.00101

Tube 4

.211

.0070

7.84 mM-1 cm-1

.00090

Tube 5

.114

.0038

7.84 mM-1 cm-1

.00048

Table 2 shows the rate of reaction in each tube as well as the calculated E value and velocity.

For the Michaelis-Menten Plot, we estimated the Vmax to be 0.0014 mM/min and Km to be 0.025 mM. In this plot, we can see the hyperbolic relationship between the velocity of the reaction and the concentration of PNPP. Km and Vmax were determined solely from the graph without calculations meaning that these values will be the least accurate of all the plots (See figure 1).

Figure 1 showing velocity vs the concentration of PNPP.

For the Lineweaver-Burk plot, we found the Vmax and Km by using the equation of the line from the plot. We solved for Vmax by using the y-intercept which is 1/vmax giving us a value of 0.0016 mM/min and to get the Km we multiplied the slope and Vmax which gave us 0.027 mM (See figure 2).

Figure 2 shows the inverse of velocity vs. the inverse of the PNPP concentration.

For the Eadie-Hofstee plot, we found the Vmax and Km by again using the equation of the line from the plot. The Vmax in this equation is equal to the y-intercept which in our equation came out to 0.0015 mM/min and to get the Km we took the absolute value of the slope which gives us .021 mM (See figure 3).

Figure 3 shows velocity vs. velocity over the concentration of PNPP.

For the Hanes-Woolf plot, we found the Vmax and Km by once again using the equation of the line from the plot. To find the Vmax, we used the slope which is 1/Vmax which gave us the value 0.0015 mM/min and to get the Km we multiplied the Vmax with the y-intercept which gives us 0.024 mM (See figure 4).

Figure 4 shows the substrate concentration over velocity vs. substrate concentration.

Km and Vmax Values From Plots Plot Km (mM) Vmax (mM/min) Michaelis-Menten 0.025 0.0014 Lineweaver-Barke 0.027 0.0016 Eadie-Hofstee 0.021 0.0015 Hanes-Woolf 0.024 0.0015 Table 3 shows the Km and Vmax values for all four graphs generated from the assay results. To calculate our Kcat and Kcat/Km values, we used our Eadie-Hofstee plot because the km and vmax values were the closest to the literature values. We found our Kcat value to be 7.4 sec^-1 which is significantly lower than the literature value of 12 sec^-1. We also determined that our kcat/km was 352.38 1/mM*sec which is much lower than the literature value of 33,000 1/mM*sec. This low Kcat and low kcat/km value corresponds to a low turnover rate and low catalytic efficiency. This could be due to storing our enzyme in a cold room over the six week duration of our experiment. From the Dixon plot, we determine our Ki to be 0.0052 and by comparing to the literature value, the literature value being 0.0011-0.065 mM, we can conclude that our value is within the range. This indicates that our enzyme had a higher affinity for the Pi inhibitor.

Figure 5 shows the dixon plot of the inverse of the velocity vs. the concentration of the inhibitor Pi.

References 1. Ninfa, Alexander J., and David P. Ballou. Fundamental Laboratory Approaches for Biochemistry and Biotechnology. John Wiley & Sons, 2015....