Bovine pancreatic ribonuclease A is an enzyme that digests RNA. It is very stable. It retains its ac — Physical Chemistry — Thermodynamics Chemistry Question
Ribonuclease
Bovine pancreatic ribonuclease A is an enzyme that digests RNA. It is very stable. It retains its activity after heating in water at 100 °C and pH = 7, while practically all other enzymes are inactivated by that treatment. The stability of ribonuclease A is attributed to an unusually stabile 3D-structure kept together by four S-S bridges between its eight cysteine residues. The S-S bridges are formed by oxidation of the thiol groups present in the cysteine according to the equation: [VISUAL]
Reducing agents such as 2-mercaptoethanol can cleave the S-S bridges. Complete the equation for the cleavage using two equivalents of 2-mercaptoethanol. Draw the structures A, B and C. [VISUAL]
Model Answer
[VISUAL]
Which other factors are known to determine the 3D structure of a protein?
[ ] high proline content,
[ ] atmospheric pressure,
[ ] electrostatic forces,
[ ] gravity,
[ ] hydrogen bonds,
[ ] magnetic forces,
[ ] the size of the organism (large animals have more stable proteins),
[ ] van der Waals forces.
Mark your answer. More than one answer may be correct.
Model Answer
Electrostatic forces, hydrogen bonds and van der Waals forces.
Treatment of ribonuclease A a solution of urea (c = 8 mol dm–3 ), H2N-CO-NH2, in the presence of 2-mercaptoethanol (c = 0.01 mol dm–3 ) results in a complete disappearance of the enzymatic activity by disruption of S-S bridges. Slow removal of the urea and 2-mercaptoethanol by means of dialysis together with re-oxidation in the presence of oxygen restores the enzymatic activity. The classical experiment carried out by Christian Anfinsen nearly fifty years ago was interpreted as the proof that proteins spontaneously fold into their native, biologically active 3d-conformation. In a modified experiment Anfinsen only removed the 2-mercaptoethanol and brought it, still in presence of the urea solution, in contact with oxygen. Now S-S bridges were formed at random. Subsequent removal of urea led to the restoration of approximately 1 % of the enzymatic activity.
Assume that only one specific set of S-S bridges out all possible combinations renders the protein enzymatically active. Assume also that every possible combination of S-S bridges gas an equal probability of being formed under experimental conditions described.
Calculate the resulting fraction of enzymatically active ribonuclease A.
Model Answer
There are 8 Cys residues. The probability that any residue is coupled to its correct partner is 1 : 7. Next there remain 6 residues to consider. The chance that any of those is coupled to its correct partner is 1 : 5, etc. Therefore the fraction of active molecules is: 1/7 × 1/5 × 1/3 × 1/1 = 1/105.