NMR studies of the mechanism of inhibitors of serine proteases /
| Main Author: | |
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| Other Authors: | , , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1990.
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| Subjects: | |
| Online Access: | ProQuest, Abstract Link to OAKTrust copy |
| Abstract: | Two peptide aldehydes, leupeptin (Ac-Leu-Leu-Arg-CHO) and N-benzoyl phenylalaninal, were synthesized with 13C labels at the aldehyde carbon in order to study the interaction of these transition state analogs with serine proteases trypsin and chymotrypsin, respectively. 13C and 1H NMR chemical shift data for the two complexes formed between the Ser 195 O[gamma] of the serine proteases and the 13C-enriched aldehyde of the inhibitors reveal the presence of a hemiacetal. The NMR data also showed the formation of two diastereomeric forms corresponding to R and S configuration at the new asymmetric center created. The signals corresponding to R and S configurations of the resultant hemiacetal carbon are pH dependent. The chemical shift of the signals changed with pH following a titration curve with a pK[a] of 4.7 for the trypsin-leupeptin adduct and 6.6 for the chymotrypsin-benzoyl phenylalaninal. The proton 13C-edited NOE studies indicate a preference of the newly formed tetrahedral center, in the trypsin-leupeptin complex, for the S configuration at pH 7, or residence of the oxyanion in the oxyanion hole of trypsin. The R configuration involving H-bonding of the hydroxyl hemiacetal to His 57 is favored at pH 3. Two N-substituted pentapeptide inhibitors (Leu-Leu-Arg-Sar-Tyr and Leu-Leu-Arg-Pro-Tyr) were synthesized with a 13C label at the arginine carbonyl which is the residue that interacts with the Ser 195 O[gamma] of trypsin. We expected that the substitution on the nitrogen of the scissile bond would slow the hydrolysis reaction enough to detect the tetrahedral intermediate. The two pentapeptides Leu-Leu-Arg-Sar-Tyr and Leu-Leu-Arg-Pro-Tyr show slow hydrolysis at different rates, but while the rate of hydrolysis could be followed by NMR, no signals in the 90-110 ppm region corresponding to tetrahedral intermediates were present. A new 1H NMR technique was developed to detect the hydrogen attached to the 13C labeled carbon of aldehyde inhibitors. The proton NMR confirmed the 13C NMR observation of the hemiacetal. This technique was improved further producing a semi-selective 13C edited proton NOE method for analysis of the environment around the hydrogen of 13C labeled carbon, which made it possible to determine unequivocally the identity of the S isomer (trypsin-leupeptin complex) at high pH. |
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| Item Description: | Typescript (photocopy). Vita. "Major subject: Chemistry." |
| Physical Description: | xvi, 128 leaves : illustrations ; 29 cm |
| Bibliography: | Includes bibliographical references. |