Mutant OPAA enzyme with increased catalytic efficiency on organophosphorus compound GP
Inventors
Harvey, Steven P • Dixon, Melissa M • Guelta, Mark A • McMahon, Leslie R
Assignees
United States Department of the Army • Government of the United States of America
Publication Number
US-10335465-B1
Publication Date
2019-07-02
Expiration Date
2038-01-26
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Abstract
The invention is directed toward mutant, non-wild-type organophosphorus acid anhydrolase enzymes having three site mutations, methods of production, and methods of use to effectively degrade toxic organophosphorus compounds, most preferably GP (2,2′-dimethylcyclopentyl methylphosphonofluoridate).
Core Innovation
The invention relates to mutant, non-wild-type organophosphorus acid anhydrolase enzymes having three site mutations at sequence positions 212, 342, and 215. These mutations enhance catalytic efficiency for degrading the toxic organophosphorus compound GP (2,2′-dimethylcyclopentyl methylphosphonofluoridate). The mutant OPAA enzyme variants are produced and used for catalytic degradation of GP, providing improved detoxification methods compared to wild-type enzymes.
The problem being solved addresses the lack of an efficient and easily produced catalyst capable of degrading the highly toxic nerve agent GP. While native wild-type OPAA enzymes possess some catalytic activity against various organophosphorus nerve agents, their activity against GP is limited and marginally useful for decontamination or treatment. The invention overcomes this limitation by introducing specific amino acid substitutions that significantly increase the enzyme's catalytic efficiency on GP.
Claims Coverage
The patent includes three independent claims focused on the mutant enzyme, methods of degrading GP using the mutant, and kits containing the mutant enzyme.
Mutant organophosphorus acid anhydrolase enzyme
A mutant OPAA enzyme comprising the amino acid sequence of SEQ ID NO: 2, which includes substitutions at positions 212, 215, and 342 that enhance catalytic efficiency toward GP.
Method of degrading GP using the mutant enzyme
A method of degrading GP by contacting it with a mutant organophosphorus acid anhydrolase comprising the amino acid sequence of SEQ ID NO: 2.
Kit for degrading GP containing mutant enzyme and carrier
A kit comprising the mutant OPAA enzyme with the amino acid sequence of SEQ ID NO: 2, together with a pharmaceutically-acceptable carrier, optionally including adjuvants or excipients.
The independent claims cover the mutant enzyme with three specific mutations for enhanced GP degradation, methods utilizing this enzyme to degrade GP, and kits comprising the enzyme and carrier for practical use.
Stated Advantages
The mutant OPAA enzyme exhibits approximately four times greater catalytic efficiency on GP compared to the wild-type enzyme.
The mutant's kcat value is about twice that of the wild-type, and its Km is about half, indicating higher enzymatic efficiency and substrate affinity.
The mutations narrow the enzyme's small substrate-binding pocket, restricting GP orientation to one favorable for catalysis, enhancing degradation effectiveness.
Documented Applications
Use of the mutant OPAA enzyme for in vivo treatment of GP poisoning in subjects.
Catalytic decontamination of GP from surfaces or environmental sources.
Pharmaceutical compositions containing the mutant OPAA for administration via multiple routes including intravenous, subcutaneous, and intraperitoneal injection.
Use of kits comprising the mutant OPAA and pharmaceutically acceptable carriers or adjuvants for practical therapeutic or decontamination applications.
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