p.A199S/F227A/S287G/A328W/Y332G/F364C/M532C Ala199Ser/Phe227Ala/Ser287Gly/Ala328Trp/Tyr332Gly/Phe364Cys/M532Cys (p.A227S/F255A/S315G/A356W/Y360G/F392C/M560C Ala227Ser/Phe255Ala/Ser315Gly/Ala356Trp/Tyr360Gly/Phe392Cys/Met560Cys in primary sequence with 28 amino-acids signal peptide) denoted E364-532 for convenience. Mutations F364C/M532C introduce a new disulfide cross-subunit bond on the BChE mutated for increased cocaine hydrolysis
Kinetic parameters
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References:
Title: Amino-acid mutations to extend the biological half-life of a therapeutically valuable mutant of human butyrylcholinesterase Fang L, Hou S, Xue L, Zheng F, Zhan CG Ref: Chemico-Biological Interactions, 214C:18, 2014 : PubMed
Cocaine is a widely abused and addictive drug without an FDA-approved medication. Our recently designed and discovered cocaine hydrolase, particularly E12-7 engineered from human butyrylcholinesterase (BChE), has the promise of becoming a valuable cocaine abuse treatment. An ideal anti-cocaine therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long biological half-life. However, recombinant human BChE and the known BChE mutants have a much shorter biological half-life compared to the native human BChE. The present study aimed to extend the biological half-life of the cocaine hydrolase without changing its high catalytic activity against cocaine. Our strategy was to design possible amino-acid mutations that can introduce cross-subunit disulfide bond(s) and, thus, change the distribution of the oligomeric forms and extend the biological half-life. Three new BChE mutants (E364-532, E377-516, and E535) were predicted to have a more stable dimer structure with the desirable cross-subunit disulfide bond(s) and, therefore, a different distribution of the oligomeric forms and a prolonged biological half-life. The rational design was followed by experimental tests in vitro and in vivo, confirming that the rationally designed new BChE mutants, i.e. E364-532, E377-516, and E535, indeed had a remarkably different distribution of the oligomeric forms and prolonged biological half-life in rats from approximately 7 to approximately 13h without significantly changing the catalytic activity against (-)-cocaine. This is the first demonstration that rationally designed amino-acid mutations can significantly prolong the biological half-life of a high-activity enzyme without significantly changing the catalytic activity.
