6. Enterotoxinogenic Escherichia coli produce oligomeric human enterotoxins (hEt
ID: 1036247 • Letter: 6
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6. Enterotoxinogenic Escherichia coli produce oligomeric human enterotoxins (hEtx) comprising one A-subunit (27,000 Daltons) and five B-subunits (11,700 Daltons). The "B-subunits act as a carrier system, binding to GMi- ganglioside receptors found.. on the surfaces of eukaryotic cells.... The crystallographic structure of pEtx [porcine enterotoxin, which is closely related to human enterotoxin] revealed that the B-subunits are assembled into a pentameric toroid ring. Each B- subunit interacts extensively with its adjacent subunits via multiple hydrophobic, hydrogen bond, and salt bridge interactions." As a result, the B-pentamers are highly stable and are being studied as potential oral delivery systems that could shuttle molecules through the acidic stomach to the gut and other mucosa. The figure below shows an Arrhenius plot for the disassembly of hEtxB (o) at pH 1.68 and hEtxB214 (A; a mutant protein in which the C-terminal amino acid Asn-103 has been deleted, thus removing a stabilizing inter-subunit salt bridge between a C-terminal carboxylic acid and the R-group of the Lys-23 of an adjacent B subunit) at pH 2.60 2.0 3.50 3.35 103 x 3.40 3.45 1 /temperature (Ki) 3.30 Disassembly of hEtxB was shown to have a first-order rate constant that shows a linear dependence on the square of the H ion concentration with a pH-threshold for disassembly below pH 2.0. What is the value of the activation energy for disassembly of hEtxB? (2 pts) a. b. Disassembly of hEtxB214 was shown to have a first order rate constant that shows a linear dependence o1n the square of the H ion concentration with a pH threshold for disassembly below pH 3.5. What is the value of the activation energy for disassembly of hEtxB214? (2 pts) Explain the two pieces of evidence that suggest that hEtxB214 is less stable than hEtxB. (2 pts) c. d. Explain why hEtxB214 is less stable than hEtxB (e.g., What is it about its structure that makes it less stable?). (2 pts) At a temperature of 300 K and a pH conducive to disassembly of both molecules, by what -fold rate increase (e.g., khEtxB214/khEtxB) would hEtxB214 disassemble relative to hEtxB? (2 pts) e.Explanation / Answer
1. According to the Arrhenius Equation, K = A*e^(-Ea/RT) where A is the pre-exponential factor, Ea is the activation energy, R is the gas constant and T is the temperature.
2. Taking log on both sides, we get, lnk= lnA -(Ea/RT); Therefore, there is a linear relationship between lnk and (1/T). The slope of the line is -Ea/R.
A and B) Find the slope of both the lines. The slope will be a negative value, multiply it with -R to get Ea.
(C) Compare the two activation energies, you will find that the activation energy for the mutant proteinis lower than the activation energy of the protein. The lower activation energy is easily attainable, therefore, leading to a faster dissassembly of the mutant protein.
The higher pH range at which dissassembly occurs in the mutant protein also indicates that it is less stable compared to the normal protein. Note, that the rate of dissociation is directly proportional to [H+] squared.
(D) The removal of the stabilizing salt-bridge makes the mutant less stable as compared to the normal protein.
(E) Rate 1/ Rate2 = K1/K2.
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