Inhibition of chicken liver carboxylesterase by activated carbonyls and carbonyl hydrates.
Identical Kcat values (approximately 40 s-1) are obtained for the chicken liver carboxylesterase catalyzed hydrolysis of phenyl, p-nitrophenyl and o-nitrophenyl benzoates providing support for the involvement of an acyl-enzyme pathway, with the rate-limiting deacylation of a common benzoyl-enzyme intermediate. Chicken liver carboxylesterase catalyzed fragmentation of (E)-benzilmonoxime O-2,4-dinitrophenyl ether shows a pH dependence on a group active in the free base form with a pK'a approximately 5.0. The Ki-pH profile for benzil inhibition shows a dependence on a similar group with a pK'a = 5.4. The reactions between chicken liver carboxylesterase and the hydrated aldehyde, chloral hydrate, have shown this compound to be at once a substrate and potent inhibitor of the enzyme. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is first dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent aldehyde produces a hemiacetal adduct. The Ki value for chloral hydrate inhibition calculated from the kinetic analysis (90 nM) compares favourably with the value measured from the steady-state kinetics (87 nM).
Berndt
M C
MC
Department of Biochemistry, University of Queensland, Brisbane, Australia.
Bowles
M R
MR
King
G J
GJ
Zerner
B
B
D000818:Animals; D001565:Benzoates / Q000737:chemistry; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors*; D002384:Catalysis; D002645:Chickens; D002697:Chloral Hydrate / Q000737:chemistry*; D004136:Dinitrobenzenes / Q000737:chemistry; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004952:Esters / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology
D001565:Benzoates; D004136:Dinitrobenzenes; D004791:Enzyme Inhibitors; D004952:Esters; D002697:Chloral Hydrate; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase