W0406

Exploring the Catalytic Mechanism of Golgi α-Mannosidase II. Douglas A. Kuntz¹, Shin Numao², Stephen G. Withers², Mario Pinto³, and David R. Rose¹; ¹Div. of Structural and Molecular Biology, Ontario Cancer Institute, ²Dept. of Chemistry, University of British Columbia, and ³Dept. of Chemistry, Simon Fraser University.

The ability to crystallize and determine the structure of inhibitor complexes and active site mutants allows one to understand the catalytic mechanism of an enzyme. Very fine details of the mechanism can be elucidated if the data can be obtained to high resolution. A major focus of our work is Golgi alpha-mannosidase II (GMII), a crucial enzyme in the protein glycosylation pathway and a potential target for anti-metastatic drugs. Towards the goal of developing better, more specific, inhibitors we have solved the structure of the Drosophila ortholog (dGMII), and based on this structure created mutations in the potential nucleophile and acid-base catalyst residues. Through our collaboration with organic chemists we have access to inhibitors of various structural classes and potencies. We will present some of our recent results with high-resolution (to 1.2Å) data collected on crystals of native and mutant dGMII in complex with inhibitors, substrates, and covalent inactivators, in the context of developing a comprehensive picture of the enzyme's action.