| Autor: |
Hester KP; Department of Physiological Sciences , Oklahoma State University , Stillwater , Oklahoma 74078 , United States., Bhattarai K; Department of Entomology and Plant Pathology , Oklahoma State University , Stillwater , Oklahoma 74078 , United States., Jiang H; Department of Entomology and Plant Pathology , Oklahoma State University , Stillwater , Oklahoma 74078 , United States., Agarwal PK; Department of Biochemistry & Cellular and Molecular Biology , University of Tennessee , Knoxville , Tennessee 37996 , United States.; Arium BioLabs , 2519 Caspian Drive , Knoxville , Tennessee 37932 , United States., Pope C; Department of Physiological Sciences , Oklahoma State University , Stillwater , Oklahoma 74078 , United States. |
| Abstrakt: |
The single residue mutation of butyrylcholinesterase (BChE G117H ) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in K M and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity. |
