CRYSTALLOGRAPHIC STUDIES OF CATALYTIC-SITE MUTANT [[alpha]]-AMYLASE FROM BACILLUS SUBTILIS. H. Mizuno, N. Doui, Z. Fujimoto, T. Matsumoto and K. Takase. National Institute of Agrobiological Resources, Tsukuba 305, Japan

[[alpha]]-Amylase catalyzes the hydrolysis of [[alpha]]-D-(1,4)-glucosidic bond of starch or related carbohydrates. Site-directed mutagenesis of Bacillus subtilis [[alpha]]-amylase has been performed to understand the role of active site residues in catalysis¹⁾. Further understanding of catalytic mechanism could be made by the X-ray analysis using a catalytic-site mutant EQ208.

EQ208 crystalizes in space group P2₁2₁2₁, cell constants a=72.6, b=74.4, c=116.7Å. All data sets were collected at PF, Tsukuba ([[lambda]]=1.00Å). Heavy atom derivatives were obtained by soaking method. One platinum position was easily obtained from difference and anomalous Patterson maps. The Hg positions were located in a difference Fourier map made with single isomorphous replacement phases from the platinum derivative. Heavy atom positions were refined with MLPHARE using reflections from 50-3.0 Å resolution.

A multiple isomorphous replacement map was calculated and solvent-flattened with DM. The free R-factor was lowered from 0.55 to 0.365. The resulting maps allowed tracing of some helices. Interpretation of the main chain of EQ208 is in progress. Heavy atom derivative method is also in progress to get better phases.

1) Takase, K. et al. (1992) Biochim. Biophys. Acta 1120, 281-288