Halo enol lactone and protio enol lactone inhibitors of alpha-chymotrypsin: Mechanism and stereospecific behavior of inhibition
Baek, Du-Jong
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Permalink
https://hdl.handle.net/2142/22512
Description
Title
Halo enol lactone and protio enol lactone inhibitors of alpha-chymotrypsin: Mechanism and stereospecific behavior of inhibition
Author(s)
Baek, Du-Jong
Issue Date
1989
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Chemistry, Organic
Chemistry, Pharmaceutical
Language
eng
Abstract
A kinetic study of inactivation of $\alpha$-chymotrypsin by enol lactones was performed. Halo enol lactones, 3-(1-naphthyl)-6(E)-(iodomethylene)tetrahydro-2-pyranone ($\alpha$Np6I) and 3-phenyl-6(E)-(bromomethylene)tetrahydro-2-pyranone ($\alpha$Ph6Br), showed burst kinetics in the inactivation of the enzyme, which requires a complicated kinetic scheme including partitioning of the first acyl enzyme between the covalent derivatization of the enzyme by the revealed halomethyl ketone reactive group (permanent inactivation) and formation of the second acyl enzyme by the hydrolysis of the halomethyl ketone of the first acyl enzyme to the non-reactive hydroxymethyl ketone group (transient inhibition). Deacylation of the second acyl enzyme leads to reactivation to free enzyme, which will be available for inactivation again. From the rate of the time-dependent irreversible inactivation and the rate of deacylation, a partition ratio between transient inhibition and permanent inactivation was calculated. A halo enol lactone with a small partition ratio showed a large burst. The efficiency of halo enol lactones as suicide inactivators is thus limited by the hydrolysis of the halomethyl reactive group.
The corresponding protio enol lactones showed only transient inhibition (not inactivation) due to the lack of halogen in the lactone molecule, but deacylation rates of 4-phenyl-6-methylenetetrahydro-2-pyranone ($\beta$Ph6H) and 3-(1-naphthyl)-6-methylenetetrahydro-2-pyranone ($\alpha$Np6H) were very slow, and they are thus still effective inhibitors by formation of stable acyl enzymes. In order to understand this slow deacylation and stereospecific behavior of inhibition, protio enol lactones were resolved into their two enantiomers, and the effects of the structure variation of the lactones on the inhibition kinetics were studied. This study shows that S enantiomer binds more strongly to the active site of the enzyme than R enantiomer, and the deacylation rates of the R enantiomers of $\beta$Ph6H and $\alpha$Np6H are very slow, with the half-lives of 4.0 h and 12.6 h at pH 7.2, respectively. The enantioselectivity of deacylation of these lactones is very large, with the R enantiomers having 60-70 fold slower deacylation rates than the S enantiomers. This slow deacylation could be explained by a twisted acyl enzyme model. Since deacylation is the rate-determining step, these lactones with very slow deacylation rates act as alternate substrate inhibitors.
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