Structural and functional analysis of glycosyltransferase mechanisms

Insight into the biochemical mechanisms utilized by retaining and inverting glycosyltransferase enzymes is an important stepping stone to the directed design of stereospecific inhibitor based drugs. The suitability of proposed mechanisms was probed using site directed mutagenesis of catalytically relevant residues as well as the use of catalytically inactive substrate analogs UMP-PO2-CH2-D-Gal and α-L-Fuc-(1→2)-β-D-(3-deoxy)-Gal-O(CH2)5CH3 with the retaining human enzyme a specific α-1,3-N-acetylglucosaminyltransferase (GTA) in conjunction with kinetic and structural approaches including two dozen high resolution X-ray structures and a 2.5 Å resolution neutron structure. The neutron structure depicts a remarkably non-polar active site which lacks suitably positioned hydrogen atoms to support a dissociative mechanism. Site directed mutagenesis of residues which should be essential to initiate and stabilize a dissociative oxocarbenium ion do not abolish enzyme activity. The catalytically inactive substrate analogs depict the acceptor nucleophile to lay very close to the anomeric carbon (2.5 Å), which is considerably closer than the closest observed enzymatic dipoles (4.8 Å). This is an indication that the active site architecture is more suited to facilitate a mechanism initiating with nucleophilic attack than dissociation. To ensure that these observations are applicable to other glycosyltransferases, in depth geometric analysis of all published liganded structures of GT-A fold glycosyltransferase enzymes are reported that display conserved architectures in which the acceptor nucleophile approach is closer than enzymatic dipoles required for dissociation for both inverting and retaining enzymes. Inverting and retaining enzymes present the donor sugar through different conserved geometries about the divalent cation cofactor: all inverting enzymes position the donor for inline nucleophilic attack by the acceptor, the retaining enzymes position the sugar to be attacked from an orthogonal angle. Such an orthogonal associative mechanism is the most direct proposed approach, and seems supported by all available evidence.

Author dc:creator

• Schuman, Brock

Advisor dc:contributor.supervisor

• Evans, S. V.

• enzymes
• inhibitor
• mutagenesis
• resolution
• non-polar
• substrate

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/1828/4169

OAI identifier oai:identifier

oai:dspace.library.uvic.ca:1828/4169