Probing Synergy between Two Catalytic Strategies in the Glycoside Hydrolase O-GlcNAcase Using Multiple Linear Free Energy Relationships

Abstract

Human O-GlcNAcase plays an important role in regulating the post-translational modification of serine and threonine residues with beta-O-linked N-acetylglucosamine monosaccharide unit (O-GlcNAc). The mechanism of O-GlcNAcase involves nucleophilic participation of the 2-acetamido group of the substrate to displace a glycosidically linked leaving group. The tolerance of this enzyme for variation in substrate structure has enabled us to characterize O-GlcNAcase transition states using several series of substrates to generate multiple simultaneous free-energy relationships. Patterns revealing changes in mechanism, transition state, and rate-determining step upon concomitant variation of both nucleophilic strength and leaving group abilities are observed. The observed changes in mechanism reflect the roles played by the enzymic general acid and the catalytic nucleophile. Significantly, these results illustrate how the enzyme synergistically harnesses both modes of catalysis; a feature that eludes many small molecule models of catalysis. These studies also suggest the kinetic significance of an oxocarbenium ion intermediate in the O-GlcNAcase-catalyzed hydrolysis of glucosaminides, probing the limits of what may be learned using nonatomistic investigations of enzymic transition-state structure and offering general insights into how the superfamily of retaining glycoside hydrolases act as efficient catalysts.

Transition State of ADP-Ribosylation of Acetyllysine Catalyzed by Archaeoglobus fulgidus Sir2 Determined by Kinetic Isotope Effects and Computational Approaches

“…Fluorine substitution on the acetyl group causes significant rate deceleration, but not in each case 78, 79. Recent work has suggested that some of the rate deceleration observed for α-fluoro-substitution to the 2-acetamido-group could be attributable to increased positive charge on the pyranose ring at the transition state 79.…”

Nutrient-driven O-GlcNAc cycling – think globally but act locally

“…Because efforts to crystallize human OGA have not yet been successful, work to understand the catalytic mechanism of OGA has been based on structural studies of bacterial homologs (Dennis et al, 2006;He et al, 2010;Rao et al, 2006) and on mechanistic studies that use the human variant and synthetic OGA substrates and inhibitors (Ç etinbaş et al, 2006;Greig et al, 2009;Macauley et al, 2005a;Macauley et al, 2005b;Whitworth et al, 2007). These studies suggest that the catalytic mechanism in human OGA-L requires two key catalytic aspartate residues, Asp174 and Asp175, and proceeds via a two-step substrate-assisted mechanism.…”

The O-GlcNAc modification: three-dimensional structure, enzymology and the development of selective inhibitors to probe disease

“…In order to study the 'Michaelis' enzyme-substrate (ES) complex of BtGH84, we took advantage of the insight gleaned from the initial Taft analyses, above, and more recent multidimensional linear free energy relationships [51]. Thus, by using the combination of a difluoroacetyl substrate with a comparatively poor aryl leaving group, we were able to trap a Michaelis ES complex for BtGH84 [52].…”