Theoretical and experimental studies of slowly deacylating alpha-chymotrypsin acyl enzymes
Bemis, Guy William
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/20335
Description
Title
Theoretical and experimental studies of slowly deacylating alpha-chymotrypsin acyl enzymes
Author(s)
Bemis, Guy William
Issue Date
1991
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, Organic
Language
eng
Abstract
The differential deacylation rates for alpha-chymotrypsin beta-phenyl acyl enzymes 13 and 14 have been studied using the techniques of molecular mechanics and molecular dynamics. These studies indicate that the ketone side-chain of slowly deacylating acyl enzyme 13 is hydrogen bonded to the NH of Gly-216, and that the force of this hydrogen bond along with other non-bonded interactions pulls the ester carbonyl group out of the oxyanion binding hole. This distortion of geometry raises the energy of activation for hydrolysis of the acyl enzyme by providing little stabilization of the negative charge that develops during the rate determining process of tetrahedral intermediate formation. This is in contrast to the results for the relatively quickly deacylating acyl enzyme 14, which has a ketone carbonyl hydrogen bonded to the Gly-216 NH, and an ester carbonyl that remains hydrogen bonded to both NH's of the oxyanion binding hole. Additional evidence for the differential kinetics observed is provided by molecular mechanics and molecular dynamics studies of the deacylation tetrahedral intermediates formed by acyl enzymes 13 and 14. It is shown that whereas the tetrahedral intermediate formed from slowly deacylating acyl enzyme 13 has an oxyanion hydrogen bonded ideally to only the Ser-195 NH, the oxyanion formed from 14 has good hydrogen bonds to both the Gly-193 and the Ser-195 NH's.
We have probed the hydrogen bonding requirements for alpha-chymotrypsin by synthesis of a number of enol lactones and p-nitrophenyl esters designed to hydrogen bond to specific residues in the active site. p-Nitrophenyl ester 18 was designed to hydrogen bond to either the Ser-218 OH, or to the Gly-216 NH. Protio and bromo enol lactones 28, 29, 32, and 38 were designed to have a hydrogen bond with the Met-192 NH. p-Nitrophenyl esters 46 and 48 were designed to have a hydrogen bond to the Gly-216 NH and either a hydrophobic interaction with the side-chain of Met-192 (in 46), or a hydrogen bonding interaction with the oxyanion binding hole (in 48). Finally, p-Nitrophenyl esters 53 and 56 were designed to have N-acetyl groups that shield the ester carbonyl oxygen from attack by water. Indirect kinetic and chemical evidence is provided for the efficacy of these designed acyl enzymes.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
