PRELIMINARY STRUCTURE CHARACTERIZATION OF HUMAN ACID [[beta]]-GLUCOSIDASE. Feng Luo and Hengming Ke, Department of Biochemistry & Biophysics, School of Medicine, University of North Carolina, Chapel Hill 27599-7260, USA

Acid [[beta]]-glucosidase is an enzyme which hydrolyzes glucosylceramide to ceramide and glucose. Deficiency activity of the enzyme causes the accumulation of glucosylceramide in tissues, leading to a prevalent lysosomal storage disease known as Gaucher disease. The enzyme replacement therapy is the most efficient treatment of Gaucher disease, however, it is limited by its cost, at an estimate of 765,000 per patient per year. The Wall Street Journal and the New York Times called it the world most expense drug.

Our study aims at crystallization and structural characterization of the native acid [[beta]]-glucosidase, its complexes with the competitive inhibitor of N-butyl deoxynojirimycin and the transition state analogue of 2-fluoro-2-deoxyglucoside fluoride. These crystal structures will provide insight into the catalytic mechanism and help with screening the most efficient mutant for the new therapy of gene transfer. N-butyl deoxynojirimycin is specially interesting because it has been reported to inhibit the infectivity of HIV-1 and SIVmac and is currently in clinical trials for AIDS.

Crystal with a dimension 0.1 mm x 0.1 mm x 0.2 mm of the native acid [[beta]]-glucosidase has been obtained by the vapor diffusion method in the following conditions: protein concentration of 3 mg/ml, protein buffer (50 mM malaic acid, 4 mM [[beta]]-mercaptorethanol, 0.75 M ammonium phosphate, 1% glycerol, pH 6.5), reservoir buffer (50 mM malaic acid, 4 mM [[beta]]-mercaptorethanol, 1.2 M ammonium phosphate, 1% glycerol, pH 6.5), and the protein drop ( 3 ul of the protein buffer and 3 ul of the storage protein solution).