Huskerase is known to have a cysteine and an arginine in its active site and bot

Question

Huskerase is known to have a cysteine and an arginine in its active site and both are important for activity. In the proposed mechanism, the cysteine serves as a nucleophile and the arginine is involved in an electrostatic interaction important for maintenance of active site integrity. Sketch the pH profile of enzyme activity. Be sure to identify the relevant pKa values and assign them to either the cysteine or the arginine. What is the pH optimum of huskerase? You mutate the active site cysteine to a serine residue. Predict how this would impact the pH profile, providing a sketch. Justify your reasoning.

Explanation / Answer

Cysteine Pka= 8.3 and arginine Pka=12.5. The pH profile would be like a bell-shaped curve with enzyme activity on the y-axis and pH on the x-axis. Imagine on the X-axis pH value ranged between 6-14. At around pka1 = 8.3, the bell-shaped curve starts to increase and lead to the optimum pH=10.4, after that then it declines to pka2=12.5 and completes the curve.

Huskerase would have the optimum pH around 4 to 5.

Typically, oxygen and sulphur both function as nucleophiles but they causes for a minor difference in catalysis. Sulphur groups have extra d-orbital, hence they form longer bonds and gives it low Pka values. Therefore, serine is more favourable in acid-base catalysis to achieve concerted deprotonation.The low pKa of cysteine responsible for the formation of unproductive tetrahedral intermediate in contrast to original nucleophilic attack that highly favourable breakdown product. Sterically, sulphur of cysteine forms longer bonds and bulky Van der Waals radius but if it is mutated to serine, it can be trapped in unproductive orientation in the active site. Serine Pka=9.15, so that it also give shorter bell-shaped curve but starts to hike at pH=9.15 until pH=10.8 and then decline sharply.

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