Cocaine overdose and addiction are major public health problems. One novel treatment involves the administration of high activity mutants of human butyrylcholinesterase (BChE) (i.e. this would be a protein-based ‘drug') that would hydrolyze and detoxify cocaine.

Explain how His438 and Glu325 act together to generate a better nucleophile at Ser198.

The Ser198Ala, His438Ala, Glu325Ala triple mutant still catalyzes the ester hydrolysis of cocaine by a ~1000-fold over the uncatalyzed reaction.

Explain how the triple mutant enzyme can still catalyze the reaction when the catalytic triad has been ‘knocked out.'

One mutation that increases the catalytic activity of BChe is the Ala199 to Ser mutation. The hydroxyl group of Ser199 makes a hydrogen bond interaction with the carbonyl oxygen in the cocaine ester. Explain how an extra hydrogen bond to this carbonyl oxygen may increase the enzyme's catalytic rate.