Observation and catalytic promiscuity of retaining glycosidase intermediates

• Main Author: Zechel, David Louis
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/13892

This thesis is divided into two parts. The first addresses basic issues of retaining glycosidase mechanism. This includes the use of a new technique, time-resolved electrospray mass spectrometry, to directly monitor changes in the mass of Bacillus circulans |3-xylanase during its catalytic cycle. This enabled the pre-steady state kinetic parameters for the reaction it catalyses to be determined, which were in turn validated by traditional stopped-flow spectrophotometry. This study is followed by the characterization of the Cellulomonas fimi P-mannosidase (Man2A) by site-directed mutagenesis, pH-rate dependencies, Br0nsted relationships and kinetic isotope effects. The contribution of the substrate 2-hydroxyl to catalysis in Man2A is also evaluated. The second part of this thesis descibes the development of 'glycosynthases' from nucleophile mutants of Man2A, Agrobacterium sp. β-glucosidase (Abg) and Streptomyces lividans endoglucanase (CelB). When provided with a-glycosyl fluoride donors and glycoside acceptors these mutant glycosidases synthesize glycosidic bonds. In all three cases higher glycosylation activity is observed with serine nucleophile mutants than with the corresponding alanine mutants. This is ascribed to stabilization of the departing fluoride in the glycosylation transition state by the serine hydroxyl group, most likely through a hydrogen bond. Glycosidic bond cleaving activity can be restored to these nucleophile mutants by providing a small exogenous anion such as azide or formate. In addition to recovering bond cleaving activity with azide and formate, Abg and Man2A nucleophile mutants are also rescued by halides, including fluoride. This is an exceedingly rare form of enzymatic catalysis, as well as a dramatic example of how a variety of chemistries, or catalytic promiscuity, can develop from a single enzyme active site.