MIRAS PHASING OF THE CRYSTAL STRUCTURE OF THE GLYCOGEN DEBRANCHING ENZYME PROM MAMMALIAN MUSCLE. J. P. W. Petersen, E. M. Bergmann, N. B. Madsen, M. N. G. James, MRC Group in Protein Structure and Function, Dept. of Biochemistry, U of Alberta, Canada TOG 2H7
Glycogen debranching enzyme from rabbit muscle (GLIX) is a monomeric enzyme consisting of 1555 amino acids with a molecular weight of 178 KDa [1]. GLIX removes [[alpha]]-1,6 branch points in the limit dextrin left by the action of phosphorylase during the physiological degradation of glycogen. The removal of the [[alpha]]-1,6 branch points involves two different enzymatic activities. A glycosyl transferase activity which moves three glucose units from the side chain to the main chain and an [[alpha]]-1,6 glucosidase activity which removes the remaining [[alpha]]-1,6 linked glucose unit. Both activities are present in a single large subunit.
Crystals of GLIX belong to space group P21212l with unit cell dimensions a=105.0Å, b=195.0Å & c= 92.5Å and has one molecule per asymmetric unit [2]. A native data set was collected to 2.8Å. Furthermore, data were obtained for more than a dozen heavy atom derivative crystals. Currently we have obtained four useful derivative data sets: methylmercurychloride (1 and 2 sites respectively), KUO2F5 (1 site) and goldthioglucose (2 sites). The best derivative (methylmercurychloride) is isomorphous to 3.5Å (Riso=15.0 %)
Good MIRAS phases to 4.5Å allowed us to identify the solvent regions and domain structure of the crystal. Three of the derivatives have the major site in common. The only unique derivative (uranium) indicates non-isomorphism beyond 4Å resolution. Heavy atom phases to 3.4Å resolution are available and will be improved by various methods including solvent flattening, histogram matching, iterative skeletonization and envelope editing.The poster will present the current status of the structure determination of GLIX.
[1] Liu et al. Arch. Biochem. Biophys. 306, 232 (1993)
[2] Fitzgerald & Madsen. J. Crystal Growth. 76, 557 (1986)