A kinetic isotope effect and isotope exchange study of the nonenzymatic and the equine serum butyrylcholinesterase-catalyzed thioester hydrolysis.
Formylthiocholine (FTC) was synthesized and found to be a substrate for nonenzymatic and butyrylcholinesterase (BChE)-catalyzed hydrolysis. Solvent (D2O) and secondary formyl-H kinetic isotope effects (KIEs) were measured by an NMR spectroscopic method. The solvent (D2O) KIEs are (D2O)k = 0.20 in 200 mM HCl, (D2O)k = 0.81 in 50 mM HCl, and (D2O)k = 4.2 in pure water. The formyl-H KIEs are (D)k = 0.80 in 200 mM HCl, (D)k = 0.77 in 50 mM HCl, (D)k = 0.75 in pure water, (D)k = 0.88 in 50 mM NaOH, and (D)(V/K) = 0.89 in the BChE-catalyzed hydrolysis in MES buffer at pH 6.8. Positional isotope exchange experiments showed no detectable exchange of (18)O into the carbonyl oxygen of FTC or the product, formate, under any of the above conditions. Solvent nucleophile-O KIEs were determined to be (18)k = 0.9917 under neutral conditions, (18)k = 1.0290 (water nucleophile) or (18)k = 0.989 (hydroxide nucleophile) under alkaline conditions, and (18)(V/K) = 0.9925 for BChE catalysis. The acidic, neutral, and BChE-catalyzed reactions are explained in terms of a stepwise mechanism with tetrahedral intermediates. Evidence for a change to a direct displacement mechanism under alkaline conditions is presented.
Department of Chemistry and Biochemistry, California Polytechnic State University , San Luis Obispo, CA 93407.
Lori I
LI
Robins
Kristen M
KM
Meisenheimer
Emily J
EJ
Fogle
Cory A
CA
Chaplan
Richard L
RL
Redman
Joseph T
JT
Vacca
Michelle R
MR
Tellier
Brittney R
BR
Collins
Dorothea H
DH
Duong
Kathrin
K
Schulz
John F
JF
Marlier
D055162:Biocatalysis; D002091:Butyrylcholinesterase / Q000097:blood / Q000378:metabolism*; D017666:Deuterium Oxide / Q000737:chemistry*; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D010103:Oxygen Isotopes; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D004952:Esters; D010103:Oxygen Isotopes; D013438:Sulfhydryl Compounds; D002091:Butyrylcholinesterase; D017666:Deuterium Oxide