4. MBB1 is an important protein. The primary structure of MBB1 is given below:
1 M S K G E E L F T G V V P I L V E L D G D V L G G K F S V S 30
31 G E G E G D A T Y G K L T L K F I C T T G K L P V P W P T L 60
61 V T T F S Y G V Q C F
You may find the Table below useful to help you answer the following questions.
(a) Do you think MBB1 can form disulphide bonds. Explain your answer. (3 marks, 3 lines)
(b) Which residues of MBB1 could be post-translationally modified? In your answer, state the residues and the post-translational modifications. (4 marks, 4 lines)
(c) At physiological pH, what do you think the overall charge of MBB1 will be? Assume that the N-terminus and C-terminus of MBB1 are protonated and deprotonated respectively (-NH3+ and -COO-). Show your full method of working out of your answer. (5 marks, 6 lines)
The tertiary structure of MBB1 is shown below:
(d) Describe the tertiary structure of MBB1. (2 marks, 2 lines)
(e) The crystal structure of purified MBB1 found that the protein exists as a homohexameric complex. How many amino acids would you expect to find in this MBB complex? Show your full method of working out of your answer.
(2 marks, 2 lines)
5. Assuming the extinction coefficient (ε512) for Tetraphenylcyclopentadienone (TPCP) in a methanol solution is 1120 M-1cm-1, for a reaction with an change in absorbance reading of 0.75 over 4 minutes when 10 µl of 8 mg/ml enzyme has been used in an assay volume of 2 ml, calculate the specific activity in (μmoles.min-1.mg-1). (5 marks, 6 lines)
6.
Determine whether the relationship between the following can be described as linear or hyperbolic. (2 marks, 1 line per answer)
The relationship between concentration of enzyme and rate of reaction
The relationship between concentration of substrate and rate of reaction
Identify whether each of the following phrases describe competitive inhibition, non-competitive inhibition, or neither of these terms, for an enzymatic reaction.
(3 marks, 1 line per answer)
Inhibitor and substrate can bind simultaneously to the enzyme.
KM is lowered, Vmax stays the same. –
KM is increased, Vmax stays the same. –
KM remains the same, Vmax is lowered.
KM and Vmax are lowered.
Inhibitor and substrate bind to the same site.
You discover that incubation of a purified enzyme at 37ºC denatures the enzyme. However if you add substrate to your reaction at the same temperature, the enzyme remains active. Provide a feasible explanation for this observation. (2 marks, 1 line)
Two amino acids in the oxyanion hole of chymotrypsin are important for stabilization of the oxyanion intermediate. These are Ser 195 and Gly 193. Predict what might happen if either of these amino acids is mutated to alanine and provide an explanation for your answer. (3 marks, 5 lines)