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Thesis (Ph. D.)--Institute of Paper Chemistry, 1970. / Includes bibliographical references (p. 134-136).

Thesis (Ph. D.)--Institute of Paper Chemistry, 1981. / Includes bibliographical references (leaves 95-100).

Thesis (Ph. D.)--Institute of Paper Chemistry, 1957. / Includes bibliographical references (leaves 37-38).

The fungus Cunninghamella elegans has in many studies shown to have abiotransformation similar to the metabolism of mammals. If the biotransformation isgeneral, it enables the production of metabolites by the fungus and the use asreference material. The purpose of the project were to examine whether themetabolic process of C. elegans is general, with respect to the formation ofglucosides, and can be applied to different classes of drugs. During the project, theanalyses were performed on a UPLC Q-TOF, run in both MSE and MSMS mode. Themobile phase used consisted of MeOH and 0.1 % formic acid in MQ water. Toincrease the concentration of possible glucosides, the samples were subjected to anacidic or alkaline SPE. Glucosides were detected in the fungal incubates of diclofenac,buprenorphine, norbuprenorphine and oxazepam. For diclofenac, besides twodifferent glucosides (diclofenac glucoside and hydroxylated diclofenac glucoside), ahydroxylated metabolite and a hydroxylated metabolite conjugated with sulfate werediscovered. In the samples containing buprenorphine, the phase I metabolitenorbuprenorphine was also encountered. Further, in the fungal incubates ofdexamethasone a defluorinated metabolite was identified, which is a metabolicpathway never before described for C. elegans.ISSN: 1650

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2008. / Härtill 5 uppsatser.

The exoglucanase from Cellulomonas fimi catalyses the hydrolysis of cellobiose units from the non-reducing terminus of cello-oligosaccharides with overall retention of anomeric configuration. Its mechanism of action is therefore thought to involve a double displacement reaction, involving as the first step, formation of a glycosyl-enzyme intermediate (glycosylation) and as a second step, the hydrolysis of this intermediate (deglycosylation). This mechanism is investigated here through the study of the kinetics of hydrolysis of aryl β-glucosides and aryl β-cellobiosides and by employing the mechanism-based irreversible inactivators, 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2F-DNPG) and 2", 4"-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside (2F-DNPC). The study with the aryl β-glucosides revealed that this enzyme is indeed active on glucosides, a feature that had previously been undetected. A linear relationship was found to exist between the logarithm of Vmax for hydrolysis and the phenol pKa as well as between the logarithm of Vmax/Krn and me phenol pKa, showing that glycosylation is both the rate determining step and the first irreversible step for all substrates. The reaction constant calculated, ρ = 2.21, indicates a considerable amount of charge build up at the transition state of glycosylation. The linear free energy relationship study of the aryl β-cellobiosides revealed no significant dependence of the logarithm of Vmax on the pKa of the phenol, indicating that deglycosylation is rate determining. However, the slight downward trend in this Hammett plot at higher pKa values may suggest that the rate determining step is changing from deglycosylation to glycosylation. However, the logarithm of Vmax/Km does correlate with the pKa of the phenol, thus showing that the first irreversible step is glycosylation. The reaction constant (ρ = 0.60) which reflects the development of charge at the glycosylation transition state for the cellobiosides is less than that calculated for the glucosides, thus suggesting a glycosylation transition state with either a greater degree of acid catalysis or less C-O bond cleavage than that for the glucosides. The inactivators, 2F-DNPC and 2F-DNPG, are believed to inactivate the exoglucanase by binding to the enzyme and forming covalent glycosyl-enzyme intermediates. The inactivated-enzyme was stable in buffer but reactivated in the presence of a suitable glycosyl-acceptor such as cellobiose, presumably via a transglycosylation reaction. These results indicate that covalent 2F-glycosyl-exoglucanase intermediates are stable and are catalytically competent to turn over to product, thus supplying further evidence for the Koshland mechanism. The exoglucanase is inactivated more rapidly by 2F-DNPC than by 2F-DNPG. However, both inactivated forms of the enzyme reactivated at comparable rates in the presence of cellobiose, showing that the second glucosyl unit present on the cellobiosides increases the rate of glycosylation relative to that found for the glucosides but not the rate of deglycosylation. The stable covalent nature of the 2F-glycosyl-enzyme intermediates provided an excellent opportunity to identify the enzymic nucleophile. This was accomplished by radiolabelling the exoglucanase with a tritiated analogue of 2F-DNPG cleaving the protein into peptides and purifying the radiolabelled peptides. Sequencing of this peptide resulted in the identification of the active site nucleophile as glutamic acid residue 274. This residue was found to be highly conserved in this family of β-glycanases, further indicating its importance in catalysis. / Science, Faculty of / Chemistry, Department of / Graduate

The independent interconversion of alpha-N-carbobenzoxy-D-glucosaminide to the beta- form, and vice versa, suggests that the proper treatment of the rate data must involve a system of opposing reactions. It is evident from the data cited in the preceding chapter that such opposing rates must necessarily involve the HCl which is included in the reaction mixture. Thus, as the first approach to the analyzed data, it is reasonable to suppose that the rate law for opposing second-order rates will apply. However, it is also apparent from the data that the concentration of HCl used in all this work exceeds the glycoside concentration by a factor of about one hundred times. Therefore, the postulated system of opposing second order processes may be reduced to a system of opposing [pseudo-first-order processes. Such a system can be formulate as follows: [see PDF]

Some information on the hydrolysis mechanism of phosphate esters was obtained by studying the kinetics of ∝-D-Glucosamine-1-phosphate. ∝-D-Glucosamine-1-phosphate was synthesized, an experimental pH-rate profile for the hydrolysis of this phosphate was determined at 100 C. It was proposed that Species I (see page 7) had P-O cleavage with intramolecular proton transfer to ester oxygen. Species II is believed to have the same mechanism of hydrolysis as monoanion of methyl phosphate and monoanion of ∝-D-Glucosamine-1-phosphate. This study indicates that Species III s reactive, while the dianion of methyl phosphate and the dianion of ∝-D-Glucosamine-1-phosphate are inactive. The increased reactivity of Species III may be accounted for by the ability for proton transfer to ester oxygen. This investigator concluded that the amino group of ∝-D-Glucosamine-1-phosphate has an appreciable effect on the ease of P-O and C-O cleavage and the position of rate maximum.