A β-glucosidase was isolated from A. faecalis and purified 880 fold by a combination of classical and medium pressure chromatographic techniques to a specific activity of 31.6 units/mg. The protein was homogeneous by the criteria of SDS-PAGE and gel chromatography. The sub-unit molecular weight was determined to be 51,000 by SDS-PAGE. The apparent oligomeric molecular weight was determined to be 75,000 by Superose gel chromatography and 98,000 by Waters 1-250 gel chromatography, suggesting that the enzyme is a dimer. The enzyme was shown to be a retaining β-glucosidase with exo-glucanase activity only. The kinetic parameters of a number of substrates and inhibitors were determined allowing deductions to be made about the nature of the active site and catalytic mechanism. The Km's determined for cellobiose and PNPG were low for a bacterial β-glucosidase, 0.70 mM and 0.083 mM respectively. In the cellodextrin series, cellobiose to cellopentaose, the enzyme was most efficient (as defined by Vm/Km) with cellotriose as substrate. In common with other cellulolytic β-glucosidases, the glycone site showed a high specificity for glucose (although it would tolerate some modifications) and poor specificity at the aglycone site. Catalytic activity was (unusually) observed with p-nitrophenyl-β-D-mannopyranoside as substrate. Activation energies were determined by means of Arrhenius plots. / Science, Faculty of / Chemistry, Department of / Graduate

The mechanism of glucoside hydrolysis by Agrobacterium β-glucosidase has been investigated through the study of linear free energy relationships and a-secondary deuterium kinetic isotope effects. A two-step mechanism has previously been proposed for this process, consisting of; (1) cleavage of the glucosidic bond and formation of a covalent glucosyl-enzyme intermediate ("glucosylation"), and (2) hydrolysis of the intermediate to yield free enzyme and glucose ("deglucosylation"). Values of kcat and Km were determined for enzymic hydrolysis of fifteen substituted phenyl β-D-glucopyranosides with leaving group pKa's ranging from 3.96 to 10.34. A linear free energy correlation of log(kcat) vs. leaving group pKa resulted in a concave-downward plot with a break near pKa 8, indicating a change in rate-determining step of a multistep reaction. The rates of hydrolysis of substrates with leaving group pKa's < 8 are independent of phenol structure, indicating that deglucosylation is rate-limiting. Glucosides with leaving group pKa's > 8 exhibit a linear dependence of hydrolysis rate upon pKa, and thus it is proposed that glucosylation is the rate-determining step for these substrates. The value of the Hammett reaction constant, ρ, is 1.6, indicating that cleavage of the glucosidic bond is significantly advanced at the transition state. The α-secondary deuterium kinetic isotope effects on hydrolysis of five substituted phenyl β-D-glucopyranosides were determined (deuterium substitution at the anomeric center), and the values were found to be segregated into two groups. The faster substrates (leaving group pKa < 8) exhibited kH/kD values of approximately 1.11, while values for the slower substrates averaged 1.06. These results support the hypothesis of a change in rate-determining step as leaving group pKa decreases. The magnitude of the isotope effect on glucosylation indicates a small amount of sp³ to sp² rehybridization at the transitionstate, which combined with the ρ value for this process suggests a substantial degree of nucleophilic participation of the enzymic carboxylate. 2-Deoxy-2-fluoro-D-glucosides with highly activated leaving groups are potent covalent inactivators of Agrobacterium β-glucosidase which operate by trapping the enzyme as its glucosyl-enzyme intermediate. Values of ki and Ki were determined for six substituted phenyl 2-deoxy-2-fluoro-β-D-glucopyranosides whose leaving group pKa's ranged from 3.96 to 7.18. A linear correlation was observed for both log(ki) and log(ki/Ki) vs. leaving group pKa, with ρ values of 2.0 and 2.7, respectively, which indicates that cleavage of the glycosidic bond is virtually complete at the transition state. Such an observation of a linear free energy relationship between the rate of enzyme inactivation and the electronic structure of the inactivator is rarely accomplished in enzymology. Preliminary investigation of the α-secondary deuterium kinetic isotope effect on enzyme inactivation by 2',4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranoside indicates that the effect is quite small, probably 0-5%. These results suggest that the inactivation proceeds via an essentially concerted mechanism and that the transition state has little oxocarbonium ion character. / Science, Faculty of / Chemistry, Department of / Graduate

Cheung Wai-chit. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004 / Includes bibliographical references (p. 135-140) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

*Please refer to dissertation for diagrams. / In this thesis, a review concerning enantiospecific syntheses and structure activity relationship of N-linked carbadisaccharides from 1994 to 2005 is presented. / Valienamine was isolated from microbial degradation products of validoxylamine A with Pseudomonas denitrificans. It showed alpha-glycosidase inhibitory activity and antibiotic activity. Isopropylidene protected allylic chlorides, 2-epi-valienamine and 4-amino-6-deoxy-alpha- D-pseudomannopyranose were synthesized from (-)-quinic acid. The acetonide blocking groups were shown to be the best hydroxyl protecting groups for coupling precursors, compatible with palladium-catalyzed coupling reaction which afforded high yields of the 1,1'- and 1,4'-N-linked carbadisaccharides 83-88 and 89-91, respectively, with a minimum amount of an elimination diene side-product. 2-epi-Valienamines 92 and N-alkyl 2-epi-valienamine 93 and 94 were also prepared. The key palladium-catalyzed coupling reaction was shown to be a regio- and stereospecific reaction. Acid hydrolysis was used to obtain free pseudoaminosugars as glycosidase inhibitors. The inhibitory activities of these pseudoaminosugars were evaluated by the Biochemistry Department of the Chinese University of Hong Kong.* / Kwong Suk Kwan. / "January 2006." / Adviser: Tony K. M. Shing. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6409. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 146-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

by Lee Chi-Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 74-78). / Abstracts in English and Chinese. / Acknowledgment --- p.ii / Table of Contents --- p.iii / Abstract --- p.v / Abstract (Chinese Version) --- p.vi / Abbreviation --- p.vii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- General Background --- p.1 / Chapter 1.1.1 --- Valienamine --- p.2 / Chapter 1.1.2 --- Valienamine Derivatives --- p.4 / Chapter 1.2 --- Mechanistic Aspects of Glycosidase Inhibition --- p.6 / Chapter 1.2.1 --- General Background --- p.6 / Chapter 1.2.2 --- Mechanism of Enzyme Catalyzed Hydrolysis of Glycosides --- p.6 / Chapter 1.2.3 --- Types of Glucosidase Inhibitors --- p.7 / Chapter 1.2.4 --- Inhibition of Glycosidases by Valienamine Derivatives --- p.8 / Chapter 1.3 --- Previous Synthesis of Valienamine --- p.9 / Chapter 1.3.1 --- Enantiospecific Synthesis of Valienamine by Vasella and co-workers --- p.9 / Chapter 1.3.2 --- Enantiospecific Synthesis of Valienamine by Tatsuta and co-workers --- p.10 / Chapter 1.3.3 --- Synthesis of N-Alkyl Derivatives of Valienamine --- p.12 / Chapter 1.4 --- Previous Syntheses of Valienamine-containing Pseudodisaccharides and its Diastereomers --- p.12 / Chapter 1.4.1 --- Epoxide aminolysis --- p.12 / Chapter 1.4.2 --- Condensation of amine with ketone --- p.15 / Chapter 1.4.3 --- Synthesis of pseudoaminodisaccharide by Kapp and co-workers --- p.16 / Chapter 2. --- Results and Discussion --- p.18 / Chapter 2.1 --- General Strategy --- p.18 / Chapter 2.2 --- Syntheses of coupling precursors --- p.20 / Chapter 2.2.1 --- Syntheses of protected valienamine 65 and its 2-epimer80 --- p.20 / Chapter 2.2.1.1 --- Synthesis of Diol68 --- p.20 / Chapter 2.2.1.2 --- Synthesis of Diol67 --- p.21 / Chapter 2.2.1.3 --- Synthesis of protected valienamine 65 and its 2-epimer80 --- p.22 / Chapter 2.2.2 --- Syntheses of 6-deoxyaminosugars 63 and118 --- p.24 / Chapter 2.2.2.1 --- Synthesis of benzyl ether91 --- p.24 / Chapter 2.2.2.2 --- Synthesis of β-diol103 --- p.28 / Chapter 2.2.2.3 --- Synthesis of α-alcohol107 --- p.30 / Chapter 2.2.2.4 --- Synthesis of β-diol113 --- p.31 / Chapter 2.2.2.5 --- Syntheses of amines 63 and118 --- p.33 / Chapter 2.2.3 --- Syntheses of allylic chlorides --- p.34 / Chapter 2.3 --- Syntheses of pseudoaminodissaccharides --- p.36 / Chapter 3. --- Conclusion --- p.42 / Chapter 4. --- Experimental --- p.44 / Chapter 5. --- References --- p.74 / Chapter 6. --- Appendix --- p.79 / List of spectra --- p.79