Kinetic solvent isotope effects on substrate hydrolysis catalyzed by [alpha]-chymotrypsin and N[epsilon]2-histidine-57 alkylated [alpha]-chymotrypsins /
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| Other Authors: | , , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1988.
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| Subjects: | |
| Online Access: | Link to OAKTrust copy |
| Abstract: | The kinetics for the deacylation rate of N-Cbz-L-Tyr-, N-Cbz-L-Phe-, N-Cbz-L-Trp-p-nitrophenyl esters, p-nitrophenyl 3-phenylpropanoate ester, N-Cbz-L-D-Phe-p-nitrophenyl ester, and N-Glutaryl-L-Phe-p-nitroanilide were investigated by using the proton inventory technique. The inventories for the hydrolyses of N-Cbz-L-Tyr-, N-Cbz-L-Trp-, and N-Cbz-L-Phe-p-nitrophenyl esters were all bowl-shaped which is indicative of multiple proton catalysis. These inventories were consistent with either a two- or three-proton catalytic model. Arguments favoring a three-proton model involving contributions to the kinetic solvent isotope effect by the amide protons of the oxyanion hole have been presented. N-Cbz-D-Phe-p-nitrophenyl ester and p-nitrophenyl 3-phenyl-propanoate gave proton inventories which were linear and have been interpreted in terms of one-proton catalysis. Finally, N-glutaryl-L-Phe-p-nitroanilide gave a dome-shaped inventory. To disrupt the catalytic triad amino acids (Asp-102, His-57, and Ser-195), His-57 was methylated specifically at N^ε2. This modification precludes the classical charge-relay mechanism from operating. Proton inventories for the deacylation of N-acetyl-L-Tyr- and N-acetyl-L-Phe-methyl chymotrypsin and N-Cbz-L-Tyr-methyl chymotrypsin were all bowl-shaped. The [D]V's for these reactions ranged from 3.6-4.1. These inventories are indicative of multiple proton catalysis and have been interpreted in terms of a three-proton model involving contributions to the kinetic solvent isotope effect by the amide protons of the oxyanion hole. Finally, His-57 of α-chymotrypsin was ethylated and n-propylated. The inventories for the deacylation of N-acetyl-L-Tyr-ethyl chymotrypsin and N-acetyl-L-n-propyl chymotrypsin were also bowl-shaped indicative of multiple proton catalysis. |
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| Item Description: | Typescript (photocopy). Bracketed information represented by symbol on title page. Vita. "Major subject: Chemistry." |
| Physical Description: | xix, 219 leaves : illustrations ; 29 cm |
| Bibliography: | Includes bibliographical references. |