MS09-P08 pH dependent conformational changes of b-amylase/glucose complex crystal measured at room temperature Bunzo Mikami (Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan) Xueni Hou (Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan) Kotaro Mori (Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan) Adachi Motoyasu (Japan Atomic Agency, Tokai-mura, Ibaraki, Japan) Kimihiko Mizutani (Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan) Nobuyuki Takahashi (Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan)email: mikami@kais.kyoto-u.ac.jpβ-Amylase catalyzes the liberation of maltose from the non-reducing ends of starch. In contrast to a-amylase, b-amylase produces β-anomeric maltose, and is classified as an inverting enzyme. In soybean β-amylae (SBA), the hydrolysis of the α-1, 4-glycosyl linkage is proceeded by two catalytic residues, Glu186 (acid) and Glu380 (base) where the substrate binding site consists of five subsites (-2, -1, +1, +2 and +3). Near this active site, the enzyme has two mobile loops, flexible loop (residue 96-103) and inner loop (residue 341-345). The conformation of these loops change from open to closed form and from apo to product form, respectively, during enzyme action. The side-chain of Lys295 also changes conformation from apo to complex form. In this paper, we are intended to determine the structural changes of SBA/G1 (glucose) complexes in a different pH media. We have determined the crystal structure at room temperature to avoid the undesirable effect of freezing and cryo-protectant. SBA was crystallized by a hanging-drop vapor diffusion against 1 ml of the bottom solution containing 45% saturated ammonium sulfate, at pH 5.4 and 4˚C. The obtained crystals were packed in glass capillaries after soaked with 0~300mM G1 in the different pH media for 30min at 20˚C. The diffraction data sets were collected   at 20˚C with a MX225HE (Rayonix) detector at BL26B1 beam-line in SPring-8. The crystal belonged to P3121 with cell dimensions of a = b = 84-85 and c = 144-145 Å. The crystal data were collected with 98-100 % completeness and Rmerge of 0.04-0.07 up to 1.6-1.9 Å resolution. The models were refined with SHELXL with R = 0.13-0.14 and Rfree = 0.15-0.17. At pH 3.95, two G1 molecules were located at the subsites -2 and +2 with open flexible loop, apo inner loop and apo form of Lys295, whereas at pH 7.9, three G1 molecules were found at subsites -2, +1 and +2 with closed flexible loop, product form of the inner loop and complex form of Lys295 (Fig. 1). These results indicate that the conformational change of the inner loop and the side-chain of Lys295 depend on the G1 binding at +1 site. We are now trying to determine the dissociable residue controlling the sugar binding and the conformational changes of the active site.References:

Keywords: β-amylase, enzyme reaction, enzyme/substrate complex