Question

BACKGROUND The first part of this assignment focuses on the thermodynamics of enzyme-catalysed reactions. To answer the questions posed you will need apply the following two equations. Note that you may need to re- arrange these equations to determine the value you need. You also need to take care with the units of the values you use and determine. The first equation defines the relationship between the change in free energy (AG ") and the equilibrium between the products and substrates of the reaction (K'ea) under standard conditions (25°C; 1 atm; pH 7.0). Δ0"--RTInK'e Equation 1 where R is the gas constant (8.3145 J.K,mol-1) and T is the temperature in Kelvin (0°C-273%) The second equation relates the relative concentrations of products and substrates, and the temperature, to the actual free energy change (AG) Where square parentheses (I) indicate concentration, A & Bare substrates, and C&D are products. The last expression in this equation Ala cn also be referred to as the mass action ratio (Q). AlB] Note that in this example we have two products and two substrates-this may not always be the case and Q can be derived from any number of products and substrate. This assignment will focus on three enzyme-catalysed reactions found in most cells of the body. Question 3-Determining Kd (20 marks) 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] (HM) Trial 1Trial 2 Trial 3 Trial 1Trial 2 Trial 3 109.06 9.08 9.12 11.65 11.84 11.78 57.71 7.68 7.67 9.48 9.519.58 3.33 6.656.66 6.71 8.358.33 8.32 2.55.91 5.86 5.88 7.387.457.41 1.67 4.384.37 4.29 6.63 6.68 6.72 1.253.753.76 3.816.26 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 cFor 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

GIVENTHAT :-

from Above questions:

d:-

Answer:-

Negative aspect: Relying on double reciprocal plot for extraction of kinetic constants is error prone.
Positive aspect: Vmax and Km can be estimated from the plot.

e:-

Answer:

There are 2 binding sites for ATP in a single subunit of PFK1.

f :-

Answer:

Effect of citrate on the rate of reaction catalyzed by PFK 1 is that citrate allosterically inhibits PFK1. If the citrate concentration becomes high, it in turn slows down the rate of PFK1 and also reduces rate of glycolysis.

To find Bmax with Kd =1.239

where,

B_max is the total density (concentration) of receptors in a sample of tissue. To refer to the concentration of available (free) receptors, terms B_avail or B^’_maxcan be used.

Binding potential (BP) is the ratio of B_max (receptor density) to K_D (radioligand equilibrium dissociation constant), as defined by Mintun et al. (1984):

BP=BmaxKD=receptor density×affinity.