Part:BBa_K3588003

Cocaine Esterase mutant enzyme (E172-173)

The mutant enzyme of part (part:BBa_K3588001). It is able to breakdown cocaine, as well as benzoylecogonine and cocaethylene, through hydrolysis into benzoate and ecgonine methyl ester. With a dramatically increased in vitro half life from the wildtype enzyme it is currently in phase II trials as a cocaine overdose treatment, though in our project we were intending to use it to breakdown cocaine in the sewers.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1179
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Function

The enzyme was designed in order to deal with the problem of drug overdosing, by administering an enzyme with a high catalysis rate into the bloodstream of patients who are overdosing, the effects can be rapidly reduced, however the problem posed by the usage of naturally occurring CocE is its lack of thermal stability (half life at 37°C = ~12mins), so by changing the bonding of the protein chain a more thermally stable version of the enzyme was created, thereby increasing its potential for in vivo usage, thus the current trials it is undergoing.

The enzyme CocE catalyses the breakdown of cocaine into benzoate and ecgonine methyl ester

properties

Cocaine Substrate Kinetics

K_cat = 2600 min^-1 K_M = 2.9 μM

other details

half life (at 37°C) = ~ 6hrs

design considerations

This enzyme has two major changes from the wildtype, denoted by the E172-173 notation. For more on the details of the mutations and the methods that were used can be found in the paper: 'Rational Design, Preparation, and Characterization of a Therapeutic Enzyme Mutant with Improved Stability and Function for Cocaine Detoxification'