Question

The PFK1 enzyme is regulated by a number of factors in the cell, including the level of ATP. PFK1 is a homo- tetramer (four identical subunits) and ATP binds to multiple sites. The binding of ATP can be measured using radio-labelled ATP. The data presented below represents the triplicate determination of the binding of ATP to PFK in the absence and presence of 300 HM citrate Moles of bound ATP per mole enzyme -citrate +citrate [ATP] (uMTrial 1Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 10 9.06 9.08 9.1211.65 11.84 11.78 5 7.71 7.68 7.67 9.48 9.519.58 3.336.65 6.666.71 8.35 8.33 8.32 2.5 5.91 5.865.88 7.38 7.45 7.41 1.67 4.384.374.296.63 6.68 6.72 1.25 3.75 3.76 3.81 6.26 6.19 6.31 a) Determine the mean and standard deviation of the data for each concentration of ATP for both - citrate and +citrate. Show these in a table 2 marks b) Create a SINGLE double-reciprocal plot to determine the binding parameters for ATP with and without citrate present. Ensure that each dataset is distinct (use alternate colours and/or shapes for the data points) 4 marks c) For each plot give the fitted equation, the value of Kd, and the number of binding sites for ATP per PFK1. Show these in a table. Ensure you include the units of each value. Show your working for the +citrate experiment. 6 marks d) With reference the data you plotted and Kd values you determined, explain the positive and negative aspects of using a double reciprocal plot. 3 marks e) How many binding sites are there for ATP in a single subunit of PFK1? Explain your answer. 2 marks f) What effect might citrate have on the rate of the reaction catalysed by PFK1 Explain your answer 3 marks

Answer 1

   -citrate
1. 10uM Mean 9.08 SD = 0.024
2. 5uM Mean 7.68 SD = 0.016
3. 3.33uM Mean 6.67 SD = 0.026
4. 2.5uM Mean 5.88 SD = 0.02
5. 1.67uM Mean 4.34 SD = 0.04
6. 1.25uM Mean 3.77 SD = 0.026

+ citrate

1. 10uM Mean 11.75 SD = 0.079
2. 5uM Mean 9.52 SD = 0.041
3. 3.33uM Mean 8.33 SD = 0.012
4. 2.5uM Mean 7.41 SD = 0.028
5. 1.67uM Mean 6.67 SD = 0.036
6. 1.25uM Mean 6.25 SD = 0.049

S	V -citrate	V +citrate	1/S	1/V -Citrate	1/V +Citrate
10	9.08	11.75	0.1	0.1101322	0.08510638
5	7.68	9.52	0.2	0.1302083	0.10504202
3.33	6.67	8.33	0.3003	0.149925	0.12004802
2.5	5.88	7.41	0.4	0.170068	0.13495277
1.67	4.34	6.67	0.598802	0.2304147	0.14992504
1.25	3.77	6.25	0.8	0.265252	0.16

0.3 0.25 -Citrate 0.2 1/Vo 0.15 +Citrate 0.1 0.05 0.6 0.2 0.4 0.8 1/S

0.3 0.2299x0,084:1 0.25 0.2 0.15 y0.1043 R2 0.933 841 0.1 0.05 0.8 0 0.2 0.4 0.6 Axis Title