The X-ray crystal structure of the protease subtilisin shows its active site is on the surface. It binds substrate in an extended conformation and reacts only with soluble substrates. Subtilisin has a large nonpolar pocket (the S1 pocket) to bind acyl group and a shallow crevice (the S 1' pocket) to bind one substituent of a secondary alcohol group, while the other substituent remains in solvent. The reactivity and enantioselectivity of subtilisin toward nonpolar secondary alcohol esters is low. Based on its structure, we hypothesized that subtilisin would be highly reactive if the acyl group anchored the substrate to the active site and highly enantioselective if there was a large hydrophobicity difference between alcohol substituents. / To test our first hypothesis, we show that an anchoring acyl group increases protease reactivity and extends subtilisin E to a new class of substrates, N-acyl sulfinamides. Subtilisin E did not catalyze hydrolysis of N-acetyl arylsulfinamides, but did catalyze a highly enantioselective hydrolysis of N-dihydrocinnamoyl arylsulfinamides. The N-dihydrocinnamoyl group mimics phenylalanine and thus binds the sulfinamide to the active site. We then use the 3-(3-pyridyl)propionyl group as anchoring group to increase substrate solubility. We use this group to resolve multi-gram quantities of three important compounds and isolate the enantiomers without the use of chromatography. / Next, we use different anchoring acyl groups to extend several proteases to tertiary alcohol esters. We show that a syn-pentane-like interaction destabilizes the transition state for reaction of tertiary alcohols, but that the addition of an anchor group that binds substrate to the protease stabilizes transition state and enables proteases to catalyze hydrolysis of tertiary alcohol esters. / To test our second hypothesis, we show subtilisin enantioselectivity stems from a favourable hydrophobic interaction between nonpolar substituent and S1' pocket residues and favourable solvation of polar substituent in water. The enantioselectivity of a series of secondary alcohols in water varied linearly with the difference in hydrophobicity (logP/P0) of the substituents. The larger the logP/P0 difference the higher the enantioselectivity. Consistent with our hypothesis, the enantioselectivity of subtilisin toward N-acyl arylsulfinamides is high because of the large difference in substituent hydrophobicity.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.111822 |
| Date | January 2005 |
| Creators | Savile, Christopher K. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002481034, proquestno: AAINR25249, Theses scanned by UMI/ProQuest. |
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