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Mechanistic investigations of glycosaminoglycan degrading enzymes

University of British Columbia

Glycosaminoglycans are the main structural polysaccharides of vertebrates, and represent a major barrier to the spread of both bacterial infection and tumours. The enzymes by which mammals and pathogens degrade these polysaccharides use very different mechanisms, and may represent suitable therapeutic targets. In this thesis, work is presented towards an understanding of the mechanisms of Clostridium perfringens unsaturated glucuronyl hydrolase (UGL), the second enzyme in the bacterial pathway for degradation of glycosaminoglycans, and human heparanase, the enzyme by which the abundant glycosaminoglycan heparan sulfate is remodelled. For UGL, evidence was presented for a hydration reaction scheme that had previously been proposed on the basis of crystallographic evidence. This was shown by characterisation of products formed by reaction in D₂O and 10 % methanol, and by demonstrating hydrolysis of three compounds that are only expected to be turned over by the enzyme if this reaction is correct. Investigation of the effects of substituents on the transition state stability, by measurement of a linear free-energy relationship for a series of aryl glycosides, kinetic isotope effects, and rate determination for heteroatom-substituted substrates, led to the proposal of alternate mechanisms. Attempts to verify these mechanisms were made by testing of potential inhibitors, rescue of a catalytic-residue mutant, trapping of a covalent glycosyl-enzyme intermediate, or synthesis of a potential intermediate, but without success. The mechanism that appears most likely proceeds through protonation of the substrate C4-C5 double bond, with the resulting C5 positive charge being quenched by opening of the pyranose ring to give a C5 ketone and a C1-C2 epoxide. Subsequent hydration of the ketone and opening of this epoxide reforms the pyranose ring and gives the same product as direct hydration, but through a lower energy path. For mammalian heparanase, several new potential substrates and a potential inactivator were synthesized and tested. While this work was largely unsuccessful, it indicated that optimisation of sulfation patterns without modification of the aglycone is likely a futile strategy. A redesigned aglycone was proposed, representing a new path towards the goal of studying this enzyme for its eventual use as a therapeutic target in cancer therapy.

Text

2013-06-25

Attribution-NonCommercial-ShareAlike 3.0 Unported

http://hdl.handle.net/2429/44585

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-sa/3.0/