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UBC Theses and Dissertations

The catalytic mechanism of retaining [beta]-glycosidases Vocaldo, David J.


Through the detailed characterization of the catalytic mechanisms of four different retaining β-glycosidases and in consideration of the existing literature a detailed and generally applicable catalytic mechanism is proposed for the very large class of retaining β -glycosidases. The catalytic mechanism of the family 39 β -xylosidase (XynB) from Thermoanaerobacterium saccharolyticum is found to proceed through a covalent xylosyl enzyme intermediate flanked by oxocarbenium ion-like transition states. The exo- β -hexosaminidase from Streptomyces plicatus (SpHex) is demonstrated to use a mechanism involving anchimeric assistance from the 2-acetamido moiety of the substrate. The mechanism of this enzyme and, by extension, other family 20 enzymes proceeds through a bicyclic oxazoline intermediate. The functionally related exo- β - hexosaminidase from Vibrio furnisii (ExoII) employs a double-displacement mechanism proceeding through a covalent glycosyl enzyme intermediate. Despite the differences in the nature of the catalytic nucleophile and intermediate the reactions catalyzed by both these two enzymes involve transition states that are remarkable similar to each other, being highly dissociative, with significant oxocarbenium ion character. Lastly, the nature of the contentious intermediate in the hen egg white lysozyme-catalyzed hydrolysis of glycosides is investigated. In three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl enzyme intermediate is revealed during the catalytic cycle of HEWL. The three-dimensional structure of this intermediate is also determined by X-ray diffraction analysis. These studies point to the nature of the intermediate in the hydrolysis of glycosides catalyzed by all of these enzymes. On the basis of these results, we conclude that all retaining β -glycosidases perform catalysis by the formation and subsequent breakdown of a covalent intermediate species and not by the formation of a long-lived ion pair. The covalent intermediate may either be a glycosyl enzyme, as found for most retaining glycosidases, or a bicyclic oxazoline, as found for some classes of enzymes processing sugars bearing an acetamido group at the 2-position of the substrate. This general mechanism unifies the substrate distortion proposed by Phillips, the covalent intermediate first postulated by Koshland, and the electrophilic migration of the anomeric center (C-l) along the reaction coordinate. It is consistent with both the anti-periplanar lone-pair hypothesis and the principle of least nuclear motion. Most importantly, this mechanism is supported by all experimental data including kinetic isotope effects, mass spectrometry, crystal structures and enzyme kinetics for all retaining β -glycosidases studied, to the best knowledge of the author.

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