CRYSTALLIZATION AND PRELIMINARY X-RAY STUDIES OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM Escherichia coli Y. Kai^*, T. Inoue^*, Y. Nagara^*, H. Matsumura^*, K. Izui^**, *Department of Applied Chemistry, Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan, ^**Department of Applied Botany, Faculty of Agriculture, Kyoto University, Sakyou-ku, Kyoto 606, Japan

Phosphoenolpyruvate carboxylase (PEPC+; EC4.1.1.31) from Escherichia coli catalyzes the fixation of carbonate ion to form oxaloacetate and inorganic phosphate. It is composed of four identical subunits with molecular weight of ca. 100,000 dalton. Although the primary structure of PEPC has been determined in 1984,¹ the molecular structure of PEPC has not been determined. In order to study its biological function based on the three dimensional structure, we have crystallized PEPC by using PEG4000 as precipitant,² however, the quality of the diffraction pattern was rather low. To get crystals suitable for X-ray crystallographic studies, hanging drop vapor diffusion method was examined in various conditions with variety of pH, molecular weight of PEG, and additives as control parameters. The crystals under the best conditions appeared in tetragonal bipyramidal shape with orthorhombic crystal lattice, space group I222. The unit cell parameters were determined to be a=117.9, b=250.0, and c=81.8Å (1Å= 0.1 nm). Two sets of intensity data for native PEPC were obtained by using Rigaku RAXIS-IIc system and the Sakabe's Weissenberg camera in the Photon Factory, up to 3.4Å and 2.5Å resolution, respectively. These data sets were merged with PROTEIN . Among 143,033 accepted observations up to 2.5Å resolution, 34,452 were independent reflections, the completeness of which was 66.4% with an R-merge of 10.4%. More than 20 sets of heavy-atom derivatives have been measured for the structure determination by multiple isomorphous replacement method. Among these derivatives the Hg-derivative gave an excellent difference-Patterson map. The two sites of Hg atom were refined to have a mean figure of merit of 0.40 and the phasing power of 1.75 with the Cullis's R factor of 0.67 in the 20.0 - 3.5 Å resolution range by using the program MLPHARE in the CCP4 program package. The search for other good heavy-atom derivatives and the phase improvement with the solvent-flattening method are in progress.

¹Fujita, N., Miwa, T., Ishijima, S., Izui, K. & Katsuki, H. (1984) J. biochem, 95, 909-916

²Inoue, M., Hayashi, M., Sugimoto, S., Harada, S., Kai, Y. & Kasai, N. (1989) J. Mol. Biol., 208, 509-510