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Mechanistic studies of xanthomonas manihotis b-galactosidase and the development of a rapid transglycosylation screen Blanchard, Jan E.

Abstract

The Xanthomonas manihotis /3-galactosidase (BgaX) is a 66 kDa retaining glycosidase that hydrolyzes glycosidic bonds through a double displacement mechanism involving a covalent glycosyl-enzyme intermediate. Characterization of a recombinant form of this enzyme showed that it hydrolyzes pNPGal, DNPGal, pNPFuc, pNPcu- L-Ara, pNPGalNAc, pNPGlc and pNPXyl with kinetic parameters in the range of k[sub cal] = 0.0217-36 s⁻¹ and K[sub m] = 0.050-4.3 mM. The mechanism based inactivator 2,4-dinitrophenyl 2-deoxy-2-fluorogalactopyranoside was shown to inhibit BgaX through the accumulation of a 2-deoxy-2-fluorogalactosyl-enzyme intermediate with the kinetic parameters k[sub inact] = 0.030 ± 0.004 s ⁻¹ and K; = 0.031 ± 0.005 mM. The fluorogalactosyl-enzyme intermediate was long lived, with a half life of 40 hr. Peptic digestion of this labeled enzyme and analysis by HPLC/mass spectrometry allowed the elucidation of G l u²⁶⁰ as the catalytic nucleophile involved in the formation of the glycosyl-enzyme intermediate during catalysis. Retaining glycosidases are capable of catalysing the formation of glycosidic bonds through transglycosylation to an acceptor bound in the aglycone site of the enzyme. The second part of this study involved the development of a strategy to rapidly screen compounds for their potential as acceptors in transglycosylation reactions. This methodology was based on the premise that the reactivation, or turnover, of a glycosidase trapped as a 2-deoxy-2-fluoroglycosyl-enzyme is accelerated in the presence of a compound which productively binds to the aglycone site. The approach involved incubation of samples of the fluoroglycosyl-enzyme in the presence of a number of potential acceptors, followed by monitoring of the amount of enzyme reactivated due to transglycosylation at a fixed time. Using a 96-well plate format, seven glycosidases were screened in this manner using 46 different potential acceptors. Of the glycosides tested, 16-36% were positively identified as candidates to act as good acceptors for the given enzyme. Further evaluation of relative reactivation rates with these candidates was performed by monitoring the extent of reactivation of the fluoroglycosyl-enzyme species at a series of time points. The acceptors were ranked according to the observed initial velocity of reactivation. Generally, aryl glycosides and disaccharides were the preferred acceptors. Validation of the screening strategy as a method by which to identify good acceptors was carried out by the identification of products formed from some of the positively screened acceptors in four different cases.

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