New catalytic roles for serine esterases: a 19F-NMR study of the interaction of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone with chicken liver carboxylesterase.
The reactions of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone (TDPP) with chicken liver carboxylesterase have shown that this ketone hydrate is not only a potent inhibitor of the enzyme, but also a substrate for a number of enzyme-catalyzed reactions. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is initially dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent ketone then produces a hemiketal adduct. However, the slow reactivation (by dialysis) of TDPP-inhibited enzyme indicates that the interaction with this inhibitor is more complex. At equilibrium, a dissociation constant of 2.4 pM was obtained for this interaction. 19F-NMR studies of the enzyme-TDPP complex show that after pre-equilibration, the major adduct is not the hemiketal adduct. It is proposed that this final adduct is a cross-linked adduct formed between TDPP, the active-site serine and the active-site histidine. 19F-NMR studies reveal that chicken liver carboxylesterase catalyses the cleavage of TDPP to yield either fluoride ion or trifluoroacetate, and also the benzilic acid rearrangement of TDPP to alpha-trifluoromethylmandelate. These products have also been identified in model studies of the reaction between TDPP and imidazole.
Bowles
M R
MR
Department of Biochemistry, University of Queensland, Brisbane, Australia.
King
G J
GJ
Berndt
M C
MC
Zerner
B
B
D000096:Acetone / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D001894:Borohydrides / Q000737:chemistry; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002384:Catalysis; D002645:Chickens; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004950:Esterases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D005459:Fluorides / Q000737:chemistry; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy
C102842:3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone; D001894:Borohydrides; D004791:Enzyme Inhibitors; D000096:Acetone; C025364:sodium borohydride; D004950:Esterases; C022635:serine esterase; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D005459:Fluorides